e coli research paper pdf

MINI REVIEW article

Escherichia coli as a multifaceted pathogenic and versatile bacterium.

• Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, Brazil

Genetic plasticity promotes evolution and a vast diversity in Escherichia coli varying from avirulent to highly pathogenic strains, including the emergence of virulent hybrid microorganism. This ability also contributes to the emergence of antimicrobial resistance. These hybrid pathogenic E. coli (HyPEC) are emergent threats, such as O104:H4 from the European outbreak in 2011, aggregative adherent bacteria with the potent Shiga-toxin. Here, we briefly revisited the details of these E. coli classic and hybrid pathogens, the increase in antimicrobial resistance in the context of a genetically empowered multifaceted and versatile bug and the growing need to advance alternative therapies to fight these infections.

Introduction

Escherichia coli (or E. coli ) is a Gram-negative versatile bacterium, easily found and amenable to natural and random genetic alteration. There is a vast collection of sequenced E. coli genomes which exhibit different sizes and genomic diversity among commensal and pathogens, indicating a great assortment within the same bacterial species. They comprise of non-pathogenic bacteria that may act as commensals and belong to the normal intestinal microbiota of humans and many animals. There are also pathogenic variants, divided as diarrheagenic and extraintestinal pathogens, with different pathotypes and various natural hybrid strains ( Tables 1 and 2 ). These variants can be facultative or obligate pathogens. The facultative bacteria are part of the intestinal tract and may act as opportunistic pathogens when outside of their natural habitat, causing various types of extraintestinal infections. On the other hand, intestinal obligate pathogenic variants cause infections in distinct conditions, from moderate diarrhea to more threatening cases, as lethal outcome ( Kaper et al., 2004 ; Köhler and Dobrindt, 2011 ).

Table 1 Classic E. coli pathotypes main features: extraintestinal (ExPEC) and diarrheagenic (DEC).

Table 2 Hybrid pathogenic (HyPEC) main features described.

E. coli pangenome studies indicate enormous capacity to evolve by gene acquisition and genetic modification. Besides, these genomes have a mosaic-like structure consisting of a core genome, encoding essential cellular functions, and an accessory genome with flexible strain-specific sequences. Thus, E. coli is a model well established for studying the interdependence of genome architecture and the lifestyle of bacteria ( Touchon et al., 2009 ; Dobrindt et al., 2010 ).

Based on virulence factors in E. coli genomes and phenotypic traits, the human pathotypes of diarrheagenic E. coli (DEC) are differentiated from non-pathogenic E. coli and extraintestinal pathogenic E. coli (ExPEC). The ExPEC are classified as uropathogenic E. coli (UPEC), sepsis-causing E. coli (SEPEC) and neonatal meningitis-associated E. coli (NMEC) ( Kaper et al., 2004 ). Recent pathogenomics and phenotypic classification have revisited the DEC group as nine distinct pathotypes, proposed by their differential features and the essential virulence genes defining each subgroup, such as Shiga toxin-producing E. coli (STEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely-adhering E. coli (DAEC), adherent-invasive E. coli (AIEC), and cell-detaching E. coli (CDEC) ( Kaper et al., 2004 ; Pawłowska and Sobieszczańska, 2017 ) ( Table 1 ).

Herein, we briefly describe the diversity of these classic and novel emerging E. coli pathotypes and their genetic plasticity in a multifaceted organism. The mobile genetic elements are responsible for the appearance of novel hybrid strains with distinct assortment of virulence and antimicrobial resistance traits, bringing up the urgent need to reconsider the forms of treatment for these infections.

Types of E. coli : Many Flavors Within a Single Bacterial Species

E. coli is one of the most genetically versatile microorganisms and is able to colonize and persist in several niches, both in the environment or in hosts. Commensal E. coli strains colonize the gastrointestinal tract of humans a few hours after birth, resulting in a symbiotic relationship between the microbiota and its host ( Ducarmon et al., 2019 ). However, the mechanisms by which E. coli ensures this efficient symbiosis is not well known. It could be related to its high ability to use nutrients in the colon ( Fabich et al., 2008 ; Ducarmon et al., 2019 ). Several studies have shown that competition for nutrients between microbiota and pathogens limits the colonization of the pathogens, leading to fierce competition among these microorganisms ( Lustri et al., 2017 ).

Occasionally, pathogenic E. coli cannot be distinguished from commensal E. coli , only based on specific virulence factors, as some previously described in ExPEC strains ( Köhler and Dobrindt, 2011 ). However, this scenario is changing due to sophistication and availability of molecular typing methodologies. New computational approaches bring countless important information about host-pathogen relationships, reservoir, clinical diagnoses, and novel ExPEC transmission pathways ( Johnson and Russo, 2018 ). Often, virulence genes are located in transmissible genetic elements such as genomic islands, bacteriophages, insertion sequences (ISs), integrons, plasmids, and transposons; hence, they can be easily exchanged among different bacteria ( Hacker et al., 2003 ; Dobrindt et al., 2010 ). They also carry multiple antibiotic resistance genes that have been under strong selective pressure as consequence of the extensive use of antibiotics ( Brzuszkiewicz et al., 2009 ).

Common genetic changes in E. coli genomes ensure high diversity due to the gain and loss of genes through genetic modification events. There are many strains of ExPEC that normally colonize the gut asymptomatically, as members of the intestinal microbiota. Nonetheless, only a subset of ExPEC as UPEC, SEPEC and NMEC are responsible for the vast majority of infections such as urinary tract infections, sepsis, and meningitis ( Kaper et al., 2004 ). There is a great variety of virulence factors in ExPEC strains, such as adhesins (fimbrial and non-fimbrial), siderophores, toxins, invasins, the ability to survive in serum, among others. Moreover, many of these virulence factors may occur combined within the same strain and act synergistically. Despite extra factors, the septic strains always possess at least an adherence system, an iron uptake system and genes for serum survival ( Biran and Ron, 2018 ; Johnson and Russo, 2018 ) ( Table 1 ).

The genetic evolution in E. coli pathogenesis employs horizontal transfer mechanisms within same and across similar species. Therefore, the IS, transposons and integrons may facilitate novel rearrangements within the genome, such as duplication and suppression of genes and also capture of new genes. This genetic material transit can result in greater flexibility concerning various features, such as the transition of pathogenic bacteria between humans and animals, resistance to antimicrobials, appearance of emerging pathogens due to the gain of virulence genes, increased pathogenicity, among other features ( Frost et al., 2005 ; Brigulla and Wackernagel, 2010 ; Dobrindt et al., 2010 ; Jackson et al., 2011 ; Sheppard et al., 2018 ). All these conditions may contribute to the virulence of these bacteria, like the bacteriophage importance in the pathogenesis. The horizontal transfer between different strains favors the emergence of new pathogenic strains with discrepancies in the bacteriophage repertoire affecting directly their virulence ( Manning et al., 2008 ; Ogura et al., 2009 ; Dobrindt et al., 2010 ; Jackson et al., 2011 ).

The co-evolution of bacterial genomes with plasmids, besides potential genetic and phenotypic gain may impact cellular metabolism to ensure the maintenance and stability of the plasmid ( Jackson et al., 2011 ). Many ExPEC virulence genes are encoded within plasmids, often belonging to the ColV family, which encodes colicin, serum survival factors and iron uptake systems ( Biran and Ron, 2018 ). Similarly, intestinal pathogens carry a variety of types of plasmids, associated with virulence, majorly belonging to the incompatibility group IncF, which has transfer functions ( Carattoli, 2009 ). There are virulence plasmids essential for some pathotypes of E. coli , such as pINV and pAA, respectively, in EIEC and EAEC, according to each own group features ( Kaper et al., 2004 ).

Although, all ExPEC and DEC pathotypes are not enough to fully classify all pathogenic E. coli strains, since these bacteria are so variable, allowing constant appearance of distinct hybrid-formed strains within this dynamic bacterial species. The carriage of virulence genes essential to the pathogenesis of each pathotype and the ability to adapt to different conditions allow the emergence of hybrid pathogenic E. coli (HyPEC).

Genetic Plasticity and Emergent E. coli Pathogen: HyPEC

E. coli has an astonishing facility to amend very well, replicate and disseminate. These features allowed the advent of novel HyPEC. Acquired virulence genes and novel functions appear from mutation, recombination and other genetic changes. All these genetic differences have increased the occurrence of novel hybrid and antimicrobial resistance among DEC and ExPEC ( Dobrindt et al., 2003 ; Bielaszewska et al., 2007 ; Khan et al., 2018 ).

Recently, a HyPEC strain received widespread attention after an outbreak of foodborne bloody diarrhea and hemorrhagic uremic syndrome (HUS) in Germany. This outbreak of E. coli O1O4:H4 was associated with consumption of raw fenugreek sprouts, as a hybrid EAEC strain with STEC features, like Shiga toxin presence. This HyPEC was quickly sequenced and unraveled its intricate nature, but even with a quick response and identification it was not enough to avoid 3,842 hospitalizations with many fatalities in Europe and North Africa ( Bielaszewska et al., 2011 ; Rasko et al., 2011 ). Emerging processes are responsible for the HyPEC occurrences. Herein, the combined enteroaggregative features in a rare serotype was responsible to high attachment to cells and a biofilm formation ( Navarro-Garcia, 2014 ; Ribeiro et al., 2019 ). Moreover, this strain has gained stx2 gene lambdoid phage integrated in the genome, thus it may release the Shiga-toxin. These features have increased HUS occurrence during the outbreak on this HyPEC when compared to STEC ( Muniesa et al., 2012 ).

Many distinct genetic hybrid examples are reported in E. coli , such as STEC/ExPEC O80:H2 serotype, which caused HUS and bacteremia due the presence of stx2 and eae genes from STECs and pS88-like plasmid, described in meningitis, urosepsis and avian pathogenic strains of ExPEC ( Peigne et al., 2009 ; Mariani-Kurkdjian et al., 2014 ). The STEC/UPEC strain O2:H6 serotype, a STEC with virulence genes as α-hlyA , cnf1 , and clb from UPEC that have ability to cause diarrhea and urinary tract infections ( Bielaszewska et al., 2014 ). The EPEC/ETEC strain has acquired the LEE island and encodes the LT toxin ( Dutta et al., 2015 ). The broadly reported multidrug resistant E. coli ST131 is example of highly virulent ExPEC associated with urinary and bloodstream infections. It has also acquired enteroaggregative diarrheagenic phenotype due to pAA plasmid presence ( Boll et al., 2018 ). Many others HyPEC are described as case report, but not fully characterized. Here, we have briefly sampled some of the acquired genes by these strains, their direct impact in virulence and their hybrid nature ( Table 2 ). Comparable to these HyPEC, the coined terms hybrid- and hetero-pathogenic E. coli have been recently described as new combination of virulence factors among classic E. coli groups. Together, they show differences between typical and atypical subgroups within the EAEC and EPEC pathotypes and hybrids, such as EPEC/STEC, ExPEC/EPEC and ExPEC/EAEC hybrids ( Santos et al., 2020 ). Similar to our approach here, this study shows how this topic is critical in the field.

The high prevalence of classic pathogenic E. coli and appearance of HyPEC occur via similar genetic mechanisms, which also enable bacteria to resist the presence of distinct antimicrobials. Bacteria resistant to various classes of antibiotics are related to the complex combination of intrinsic and acquired resistance genes, which may act synergistically ( Cag et al., 2016 ; Khan et al., 2018 ). Together that brings multiresistant bacteria, as an alarming factor reported worldwide in several bacterial species. WHO has prioritized studies on AMR bacteria, including Enterobacteriaceae, based on recent surveillance reports ( WHO, 2018 ).

Emerging Hybrids and Alternative Therapies

The complex combination of multidrug-resistant bacteria and emerging hybrid bacteria with intrinsic or acquired bacterial virulence factors disseminated by genetic mobility elements, the intense and inappropriate use of antibiotics have simultaneously favored the emergence of resistance to various antibiotics ( Khan et al., 2018 ). That is a special challenge to these hybrid strains, since these HyPEC gathered virulence traits and acquired antibiotic resistance, together these points raise the importance to alternative treatments. These options are crucial to reduce the use of antibiotics and the consequent increase of antimicrobial resistance. Novel therapies are urgent to replace prophylactic and treatment with antibiotics by probiotics, prebiotics, enzymatic compounds, vaccines, monoclonal antibodies, phage therapy, antivirulence compounds, among other possibilities ( Gadde et al., 2017 ).

Recently, different vaccine strategies have been used for pathogenic E. coli infection as an alternative to antibiotic therapy ( Rojas-Lopez et al., 2018 ), including vaccines with attenuated toxins ( McKenzie et al., 2007 ; Bitzan et al., 2009 ), attenuated bacterial cell ( Calderon Toledo et al., 2011 ), individual components of virulence factors such as Shiga toxin ( Liu et al., 2009 ), EspA or Intimin ( Oliveira et al., 2012 ), small peptides ( Zhang et al., 2011 ), DNA ( García-Angulo et al., 2014 ) or polysaccharides ( Ahmed et al., 2006 ; van den Dobbelsteen, 2016 ), as well detailed in the literature. Commercial vaccines have aimed the use to protect livestock, such as poultry, swine and bovine herds, against respectively to APEC, like Poulvac® E. coli , ETEC and EHEC infections ( Sadeyen et al., 2015 ; Nesta and Pizza, 2018 ). Vaccines with a modern approach and technology still are a promising strategy to protect against emergent HyPECs infections in humans and livestock.

Recent studies have revisited the phage therapy as a biological alternative, which employs strictly lytic phages uncapable of lysogenization ( Carter et al., 2012 ). Studies have demonstrated ability of phages to decrease biofilm formation in UPEC ( Chibeu et al., 2012 ), increased mice rate survival in E. coli -induced pneumonia ( Dufour et al., 2015 ). Moreover, lytic bacteriophages were used to infect and kill bacteria harboring phage-dependent conjugative plasmid to avoid emergence of multiresistant bacteria ( Ojala et al., 2013 ; Tagliaferri et al., 2019 ). The phages cocktail EcoShield™ is already commercialized (Intralytix) and it has been reported to significantly reduce the E. coli O157:H7 contamination on surfaces and food ( Abuladze et al., 2008 ; Carter et al., 2012 ). Additionally, mutual use of phages with antibiotics have emerged, with SPR02 and DAF6 phages combined with enrofloxacin have shown promising data, rescuing chickens challenged with avian pathogenic E. coli infection ( Tagliaferri et al., 2019 ).

The novel approach via antivirulence-directed compounds works disarming the pathogens’ ability to cause disease by inhibiting their virulence factors, favoring the host’s immune defenses during the bacterial clearance. These compounds do not induce bacterial resistance as antibiotics, because they disarm the pathogen, instead of directly targeting its growth. Therefore, as they are directed to specific factors for pathogenesis, they potentially reduce the selection of resistance and limit collateral damage to the microbiota. Some virulence inhibitors are effective against many pathogens, molecules such as LED209, HC102A, HC103A, Artemisinin, and Ethoxzolamide, by inhibit different two-component systems as QseBC in E. coli and other enteropathogens ( Sperandio et al., 2003 ; Rasko et al., 2008 ; Yang et al., 2014 ; Xue et al., 2015 ; Kim et al., 2020 ), Bicyclic 2-pyridones, Biaryl mannoside, Nitazoxanide and FN075, avoiding the initial bacterial adhesion; and compounds like Toxtazins A and B, Ebselen, 7086, 7812, 7832, BPT15, and BBH7, blocking toxins and secretion systems ( Payne, 2008 ; Johnson and Abramovitch, 2017 ).

The forces that shape the evolution in E. coli comprise vast repertoire, affecting genetic flexibility and excessive permissiveness to acquire and donate DNA via horizontal gene transfer. These features guarantee the spread of antibiotic resistance as well as virulence factors inherited among the various pathotypes of E. coli. The exact identification and assessment assist researchers to better understand this bacterium modification, diagnosis, public health and treatment. E. coli strains with multiple and distinct factors are probably very common but unreported, since these E. coli strains have developed many strategies to persist in different settings and successfully infect the host. These strategies result in an immense variety of microorganisms, ranging from avirulent to extremely virulent strains that can cause intestinal or extraintestinal diseases. E. coli strains have great potential for dissemination and capacity to pass along hereditary elements. Currently, these HyPEC strains are a very concerning threat that demands more studies and the development of novel treatment methods.

Author Contributions

VB: writing and organization. KM: writing. CM: writing and mentoring. All authors contributed to the article and approved the submitted version.

Financially supported by FAPESP (grants 2014/06779-2, 2018/22412-2, 2018/22042-0, and 2019/03049-7), CNPq (307418/2017-0), and “Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP-PADC. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Keywords: treatment, genetic mobility, pathogenesis, Escherichia , multiresistant

Citation: Braz VS, Melchior K and Moreira CG (2020) Escherichia coli as a Multifaceted Pathogenic and Versatile Bacterium. Front. Cell. Infect. Microbiol. 10:548492. doi: 10.3389/fcimb.2020.548492

Received: 02 April 2020; Accepted: 17 November 2020; Published: 21 December 2020.

Reviewed by:

Copyright © 2020 Braz, Melchior and Moreira. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Cristiano Gallina Moreira, [email protected]

† ORCID : Vânia Santos Braz, orcid.org/0000-0002-1389-1055 Cristiano Gallina Moreira, orcid.org/0000-0002-0689-4119

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• Published: February 2004

Pathogenic Escherichia coli

• James B. Kaper 1 , 2 ,
• James P. Nataro 1 , 3 &
• Harry L. T. Mobley 2  

Nature Reviews Microbiology volume  2 ,  pages 123–140 ( 2004 ) Cite this article

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In addition to being an important member of the normal intestinal microflora of humans and other mammals, the species Escherichia coli contains many pathotypes that cause a variety of diseases. At least six different pathotypes cause enteric disease, such as diarrhoea or dysentery, and other pathotypes cause extra-intestinal infections, including urinary tract infections and meningitis.

Virulence factors of E. coli can affect a wide range of eukaryotic cellular processes, including cell signalling, ion secretion, protein synthesis, mitosis, cytoskeletal function and mitochondrial function.

Virulence factors of pathogenic E. coli are frequently encoded on genetic elements such as plasmids, bacteriophage, transposons and pathogenicity islands that can be mobilized into different strains to create novel combinations of virulence factors.

The genomic structure of the E. coli pathotypes that have been sequenced so far show a striking mosaic pattern, with 2,000 genes present in 247 islands in one pathotype that are not present in K-12. Up to 0.53 MB of DNA present in K-12 can also be absent from pathogenic E. coli .

Genes that encode virulence factors of pathogenic E. coli are regulated by both pathotype-specific regulators that are absent from commensal E. coli , and by 'housekeeping' regulators that are present in commensal E. coli .

Few microorganisms are as versatile as Escherichia coli . An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.

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Escherichia coli typically colonizes the gastrointestinal tract of human infants within a few hours after birth. Usually, E. coli and its human host coexist in good health and with mutual benefit for decades. These commensal E. coli strains rarely cause disease except in immunocompromised hosts or where the normal gastrointestinal barriers are breached — as in peritonitis, for example. The niche of commensal E. coli is the mucous layer of the mammalian colon. The bacterium is a highly successful competitor at this crowded site, comprising the most abundant facultative anaerobe of the human intestinal microflora. Despite the enormous body of literature on the genetics and physiology of this species, the mechanisms whereby E. coli assures this auspicious symbiosis in the colon are poorly characterized. One interesting hypothesis suggests that E. coli might exploit its ability to utilize gluconate in the colon more efficiently than other resident species, thereby allowing it to occupy a highly specific metabolic niche 1 .

However, there are several highly adapted E. coli clones that have acquired specific virulence attributes, which confers an increased ability to adapt to new niches and allows them to cause a broad spectrum of disease. These virulence attributes are frequently encoded on genetic elements that can be mobilized into different strains to create novel combinations of virulence factors, or on genetic elements that might once have been mobile, but have now evolved to become 'locked' into the genome. Only the most successful combinations of virulence factors have persisted to become specific ' PATHOTYPES ' of E. coli that are capable of causing disease in healthy individuals. Three general clinical syndromes can result from infection with one of these pathotypes: enteric/diarrhoeal disease, urinary tract infections (UTIs) and sepsis/meningitis. Among the intestinal pathogens there are six well-described categories: enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC) 2 ( Fig. 1 ). UTIs are the most common extraintestinal E. coli infections and are caused by uropathogenic E. coli (UPEC). An increasingly common cause of extraintestinal infections is the pathotype responsible for meningitis and sepsis — meningitis-associated E. coli (MNEC). The E. coli pathotypes implicated in extraintestinal infections have recently been called ExPEC 3 . EPEC, EHEC and ETEC can also cause disease in animals using many of the same virulence factors that are present in human strains and unique colonization factors that are not found in human strains ( Table 1 ). An additional animal pathotype, known as avian pathogenic E. coli (APEC), causes extraintestinal infections — primarily respiratory infections, pericarditis, and septicaemia of poultry. This review will focus on E. coli strains that are pathogenic for humans.

The six recognized categories of diarrhoeagenic E. coli each have unique features in their interaction with eukaryotic cells. Here, the interaction of each category with a typical target cell is schematically represented. These descriptions are largely the result of in vitro studies and might not completely reflect the phenomena that occurs in infected humans. a | EPEC adhere to small bowel enterocytes, but destroy the normal microvillar architecture, inducing the characteristic attaching and effacing lesion. Cytoskeletal derangements are accompanied by an inflammatory response and diarrhoea. 1. Initial adhesion, 2. Protein translocation by type III secretion, 3. Pedestal formation. b | EHEC also induce the attaching and effacing lesion, but in the colon. The distinguishing feature of EHEC is the elaboration of Shiga toxin (Stx), systemic absorption of which leads to potentially life-threatening complications. c | Similarly, ETEC adhere to small bowel enterocytes and induce watery diarrhoea by the secretion of heat-labile (LT) and/or heat-stable (ST) enterotoxins. d | EAEC adheres to small and large bowel epithelia in a thick biofilm and elaborates secretory enterotoxins and cytotoxins. e | EIEC invades the colonic epithelial cell, lyses the phagosome and moves through the cell by nucleating actin microfilaments. The bacteria might move laterally through the epithelium by direct cell-to-cell spread or might exit and re-enter the baso-lateral plasma membrane. f | DAEC elicits a characteristic signal transduction effect in small bowel enterocytes that manifests as the growth of long finger-like cellular projections, which wrap around the bacteria. AAF, aggregative adherence fimbriae; BFP, bundle-forming pilus; CFA, colonization factor antigen; DAF, decay-accelerating factor; EAST1, enteroaggregative E. coli ST1; LT, heat-labile enterotoxin; ShET1, Shigella enterotoxin 1; ST, heat-stable enterotoxin.

The various pathotypes of E. coli tend to be clonal groups that are characterized by shared O (lipopolysaccharide, LPS) and H (flagellar) antigens that define SEROGROUPS (O antigen only) or SEROTYPES (O and H antigens) 2 , 4 . Pathogenic E. coli strains use a multi-step scheme of pathogenesis that is similar to that used by other mucosal pathogens, which consists of colonization of a mucosal site, evasion of host defences, multiplication and host damage. Most of the pathogenic E. coli strains remain extracellular, but EIEC is a true intracellular pathogen that is capable of invading and replicating within epithelial cells and macrophages. Other E. coli strains might be internalized by epithelial cells at low levels, but do not seem to replicate intracellularly.

Adhesion/colonization. Pathogenic E. coli strains possess specific adherence factors that allow them to colonize sites that E. coli does not normally inhabit, such as the small intestine and the urethra ( Table 1 ). Most frequently these adhesins form distinct morphological structures called fimbriae (also called pili) or fibrillae, which can belong to one of several different classes ( Fig. 2 ). Fimbriae are rod-like structures of 5–10 nm diameter that are distinct from flagella. Fibrillae are 2–4 nm in diameter, and are either long and wiry or curly and flexible 5 . The Afa adhesins that are produced by many diarrhoeagenic and uropathogenic E. coli are described as afimbrial adhesins, but in fact seem to have a fine fibrillar structure that is difficult to visualize 6 . Adhesins of pathogenic E. coli can also include outer-membrane proteins, such as intimin of UPEC and EHEC, or other non-fimbrial proteins. Some surface structures trigger signal transduction pathways or cytoskeletal rearrangements that can lead to disease. For example, the members of the Dr family of adhesins that are expressed by DAEC and UPEC bind to the DECAY-ACCELERATING FACTOR (DAF, also known as CD55), which results in activation of phosphatidylinositol 3-kinase (PI-3-kinase) and cell-surface expression of the major histocompatibility complex (MHC) class I-related molecule MICA 7 . The IcsA protein of EIEC nucleates actin filaments at one pole of the bacterium, which allows it to move within the cytoplasm and into adjacent epithelial cells on a 'tail' of polymerized actin 8 . Even surface structures that are present on commensal E. coli strains can induce signalling cascades if the organism encounters the appropriate receptor. The LPS of E. coli and other Gram-negative bacteria binds to Toll-like receptor 4 ( TLR4 ), triggering a potent cytokine cascade that can lead to septic shock and death 9 . Flagellin, the main component of flagella, can bind to TLR5 , thereby activating interleukin (IL)-8 expression and an inflammatory response 10 .

E. coli produce a variety of colonization factors, many of which are hair-like structures of various morphologies called fimbriae (also called pili) or fibrillae. a | Long, straight colonization factor antigen (CFA)/III fimbriae of ETEC (5–7 nm in diameter) protruding peritrichously from the bacterial surface. b | Abundant long, straight CFA/I fimbriae (5–7 nm) of ETEC contrasting with thicker, wavy flagella. c | P pili of UPEC showing the thin ( ∼ 3 nm) fibrillar adhesive tip at the end of the pilus ( ∼ 10 nm). d | Thin (2–3 nm), flexible, wiry CS3 fibrillar structures produced by ETEC that extend several micrometres from the cell surface. e | Bundle-forming pilus (BFP) of EPEC, a member of the type IV pili family, aggregates laterally to form large rope-like structures (>10 μm long) of variable width. f | Thin (2–5 nm), coiled, highly aggregative curli fibres produced by a variety of pathogenic and non-pathogenic E. coli . Additional characteristics of colonization factors of diarrhoeagenic E. coli have been reviewed elsewhere (see Ref. 5 ). Panels a , b , d – f are courtesy of J. Girón. Panel c is reproduced from Ref. 147 Nature © Macmillan Magazines Ltd (1992).

Toxins. More numerous than surface structures that trigger signal transduction pathways are secreted toxins and other effector proteins that affect an astonishing variety of fundamental eukaryotic processes ( Table 2 ). Concentrations of important intracellular messengers, such as cyclic AMP, cyclic GMP and Ca 2+ , can be increased, which leads to ion secretion by the actions of the heat-labile enterotoxin (LT), heat-stable enterotoxin a (STa) and heat-stable enterotoxin b (STb), respectively — all of which are produced by different strains of ETEC (reviewed in Ref. 11 ). The Shiga toxin (Stx) of EHEC cleaves ribosomal RNA, thereby disrupting protein synthesis and killing the intoxicated epithelial or endothelial cells 12 . The cytolethal distending toxin (CDT) has DNaseI activity that ultimately blocks cell division in the G2/M phase of the cell cycle 13 . Another toxin that blocks cell division in the same phase, called Cif (cycle-inhibiting factor), does not possess DNaseI activity, but might act by inhibition of Cdk1 kinase activity 14 . The cytotoxic nectrotizing factors (CNF 1 and CNF 2) deaminate a crucial glutamine residue of RhoA, Cdc42 and Rac, thereby locking these important signalling molecules in the 'on' position and leading to marked cytoskeletal alterations, multinucleation with cellular enlargement, and necrosis 15 . The Map protein of EPEC and EHEC has at least two independent activities — stimulating Cdc42-dependent filopodia formation and targeting mitochondria to disrupt membrane potential in these organelles 16 .

The various toxins are transported from the bacterial cytoplasm to the host cells by several mechanisms. LT is a classic A–B subunit toxin that is secreted to the extracellular milieu by a type II secretion system 17 . Several toxins, such as Sat, Pet and EspC, are called autotransporters because part of these proteins forms a β-barrel pore in the outer membrane that allows the other part of the protein extracellular access 18 . The SPATEs (serine protease autotransporters of enterobacteriaceae) are a subfamily of serine protease autotransporters that are produced by diarrhoeagenic and uropathogenic E. coli and Shigella strains. EPEC, EHEC and EIEC contain type III secretion systems, which are complex structures of more than 20 proteins forming a 'needle and syringe' apparatus that allows effector proteins, such as Tir and IpaB, to be injected directly into the host cell 19 . The UPEC haemolysin is the prototype of the type I secretion mechanism that uses TolC for export from the cell 20 . No type IV secretion systems have been described for pathogenic E. coli , with the exception of the type IV-like systems that are involved in conjugal transfer of some plasmids. By one means or another, pathogenic E. coli have evolved several mechanisms by which they can damage host cells and cause disease.

Pathotypes and pathogenesis

Enteropathogenic E. coli (EPEC). EPEC was the first pathotype of E. coli to be described. Large outbreaks of infant diarrhoea in the United Kingdom led Bray, in 1945, to describe a group of serologically distinct E. coli strains that were isolated from children with diarrhoea but not from healthy children. Although large outbreaks of infant diarrhoea due to EPEC have largely disappeared from industrialized countries, EPEC remains an important cause of potentially fatal infant diarrhoea in developing countries 2 . For decades, the mechanisms by which EPEC caused diarrhoea were unknown and this pathotype could only be identified on the basis of O:H serotyping. However, since 1979, numerous advances in our understanding of the pathogenesis of EPEC diarrhoea have been made, such that EPEC is now among the best understood of all the pathogenic E. coli .

A characteristic intestinal histopathology is associated with EPEC infections; known as 'attaching and effacing' (A/E), the bacteria intimately attach to intestinal epithelial cells and cause striking cytoskeletal changes, including the accumulation of polymerized actin directly beneath the adherent bacteria. The microvilli of the intestine are effaced and pedestal-like structures on which the bacteria perch frequently rise up from the epithelial cell ( Fig. 3 ). The ability to induce this A/E histopathology is encoded by genes on a 35-kb pathogenicity island (PAI; see below) called the locus of enterocyte effacement (LEE) 21 . Homologues of LEE are also found in other human and animal pathogens that produce the A/E histopathology, including EHEC, rabbit EPEC (REPEC) and Citrobacter rodentium , which induces colonic hyperplasia in mice. The LEE encodes a 94-kDa outer-membrane protein called intimin, which mediates the intimate attachment of EPEC to epithelial cells 22 . Intimin not only functions as a ligand for epithelial cell adhesion, but also stimulates mucosal T H 1 IMMUNE RESPONSES and intestinal crypt hyperplasia 23 . Most of the 41 open reading frames of the core LEE PAI encode a type III secretion system and the associated chaperones and effector proteins. One of these effector proteins, known as Tir (translocated intimin receptor), is inserted into the host-cell membrane, where it functions as a receptor for the intimin outer-membrane protein 24 . This is a fascinating example of a pathogen that provides its own receptor for binding to eukaryotic cells, although additional eukaryotic proteins have also been reported to act as receptors for intimin. A recent study showed that EPEC can disrupt cell polarity, causing basolateral membrane proteins, in particular β 1 -integrins, to migrate to the apical cell surface where they can bind to intimin 25 . In addition to β 1 -integrin, Tir has also been shown to bind to NUCLEOLIN 26 . In addition to its role as a receptor for intimin, Tir has important signalling functions in epithelial cells. The portion of Tir that is exposed to the cytosol nucleates cytoskeletal proteins, initially binding directly to the adaptor protein Nck, which recruits the amino terminus of Wiskott–Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex; recruitment of Arp2/3 results in actin filament nucleation and initiation of the characteristic pedestal complex 27 ( Fig. 1 ). Interestingly, the Tir protein of EHEC O157:H7 is not functionally identical to the Tir protein of EPEC O127:H6 because pedestals are formed independently of Nck, which indicates that additional bacterial factors are translocated to trigger actin signalling 28 . Other cytoskeletal proteins, such as vinculin, cortactin, talin and α-actinin, are also recruited to the pedestal complex 29 . Formation of the pedestal is a dynamic process whereby the force of actin polymerization can propel the pedestal across the surface of ptK2 epithelial cells 30 (see movement of EPEC on ptK2 cells in the Online links). Tir also has a GAP (GTPase-activating protein) motif that has been implicated in the ability of Tir to downregulate filopodia formation 16 . Another secreted effector protein is EspF, which causes apoptosis 31 and induces redistribution of the tight-junction-associated protein occludin, which leads to loss of trans-epithelial electrical resistance 32 . As noted above, the Map protein affects mitochondrial function and filopodia formation, and additional effectors — for example, EspG and EspH — have recently been described.

The attaching and effacing histopathology results in pedestal-like structures, which rise up from the epithelial cell on which the bacteria perch. Image courtesy of J. Girón.

Additional EPEC virulence factors that are encoded outside the LEE have also been described. One very large protein of ∼ 385 kDa called lymphostatin (LifA) inhibits lymphocyte activation 33 . This protein is also present in strains of EHEC, where it is known as Efa1, and an adhesive property has been attributed to it 34 . Typical EPEC strains possess a plasmid of 70–100 kb called the EAF (EPEC adherence factor) plasmid 35 . This plasmid encodes a type IV pilus called the bundle-forming pilus (BFP) 36 , which mediates interbacterial adherence and possibly adherence to epithelial cells ( Fig. 2 ). It also contains the per locus (plasmid-encoded regulator), the products of which regulate the bfp operon and most of the genes in the LEE by the LEE-encoded regulator (Ler). So-called atypical EPEC contain the LEE but do not contain the EAF plasmid. In industrialized countries, atypical EPEC are more frequently isolated from diarrhoeal cases than are typical EPEC that contain the EAF plasmid, although typical EPEC dominate in developing countries 37 . Atypical EPEC have also caused large outbreaks of diarrhoeal disease involving both children and adults in industrialized countries.

The model of EPEC pathogenesis is considerably more complex than simple binding to epithelial cells by a single adhesin and secretion of an enterotoxin that induces diarrhoea. The emerging model, several aspects of which are reviewed elsewhere 2 , 38 , 39 , 40 , indicates that EPEC initially adhere to epithelial cells by an adhesin, the identity of which is not yet clearly established; potential candidates include BFP, the EspA filament, flagella, LifA/Efa1 and intimin (by host-cell receptors). The type III secretion system is then activated and various effector proteins — including Tir, EspF, EspG, EspH and Map — are translocated into the host cell. EPEC binds through the interaction of intimin with Tir inserted in the membrane and numerous cytoskeletal proteins accumulate underneath the attached bacteria. Protein kinase C (PKC), phospholipase Cγ, myosin light-chain kinase and mitogen-activated protein (MAP) kinases are activated, which leads to several downstream effects, including increased permeability due to loosened tight junctions. Nuclear factor (NF)-κB is activated, leading to production of IL-8 and an inflammatory response that involves transmigration of polymorphonuclear leukocytes (PMNs) to the lumenal surface and activation of the adenosine receptor. The galanin-1 receptor is upregulated 41 , thereby increasing the response of the epithelial cells to the neuropeptide GALANIN , which is an important mediator of intestinal secretion. Some, but not all, typical EPEC strains produce an enterotoxin, EspC, that increases short circuit current in USSING CHAMBERS 157 . Diarrhoea probably results from multiple mechanisms, including active ion secretion, increased intestinal permeability, intestinal inflammation and loss of absorptive surface area resulting from microvillus effacement.

Enterohaemorrhagic E. coli (EHEC). First recognized as a cause of human disease in 1982, EHEC causes bloody diarrhoea (haemorrhagic colitis), non-bloody diarrhoea and haemolytic uremic syndrome (HUS). The principal reservoir of EHEC is the bovine intestinal tract and initial outbreaks were associated with consumption of undercooked hamburgers. Subsequently, a wide variety of food items have been associated with disease, including sausages, unpasteurized milk, lettuce, cantaloupe melon, apple juice and radish sprouts — the latter were responsible for an outbreak of 8,000 cases in Japan. Facilitated by the extremely low infectious dose required for infection (estimated to be <100 cells), EHEC has also caused numerous outbreaks associated with recreational and municipal drinking water, person-to-person transmission and petting zoo and farm visitations. A recent report indicates potential airborne transmission after exposure to a contaminated building 42 . EHEC strains of the O157:H7 serotype are the most important EHEC pathogens in North America, the United Kingdom and Japan, but several other serotypes, particularly those of the O26 and O111 serogroups, can also cause disease and are more prominent than O157:H7 in many countries.

The key virulence factor for EHEC is Stx, which is also known as verocytotoxin (VT). Stx consists of five identical B subunits that are responsible for binding the holotoxin to the glycolipid globotriaosylceramide (Gb3) on the target cell surface, and a single A subunit that cleaves ribosomal RNA, causing protein synthesis to cease 12 . The Stx family contains two subgroups — Stx1 and Stx2 — that share approximately 55% amino acid homology. Stx is produced in the colon and travels by the bloodstream to the kidney, where it damages renal endothelial cells and occludes the microvasculature through a combination of direct toxicity and induction of local cytokine and chemokine production, resulting in renal inflammation (reviewed in Ref. 43 ). This damage can lead to HUS, which is characterized by haemolytic anaemia, thrombocytopoenia and potentially fatal acute renal failure. Stx also induces apoptosis in intestinal epithelial cells — a process that is regulated by the Bcl-2 family 44 . Stx was first purified from Shigella dysenteriae , and HUS can also result from infection with this species, although not with other Shigella species or EIEC, which do not produce Stx. Stx also mediates local damage in the colon, which results in bloody diarrhoea, haemorrhagic colitis, necrosis and intestinal perforation.

In addition to Stx, most EHEC strains also contain the LEE pathogenicity island that encodes a type III secretion system and effector proteins that are homologous to those that are produced by EPEC. Animal models have shown the importance of the intimin adhesin in intestinal colonization, and HUS patients develop a strong antibody response to intimin and other LEE-encoded proteins. EHEC O157:H7 is believed to have evolved from LEE-containing O55 EPEC strains that acquired bacteriophage encoding Stx 45 . Although more than 200 serotypes of E. coli can produce Stx, most of these serotypes do not contain the LEE pathogenicity island and are not associated with human disease. This has led to the use of Shiga toxin-producing E. coli (STEC) or verotoxin-producing E. coli (VTEC) as general terms for any E. coli strain that produces Stx, and the term EHEC is used to denote only the subset of Stx-positive strains that also contain the LEE. However, there are LEE-negative STEC strains that are associated with disease — for example, O103:H21 strains — thereby demonstrating that there are additional virulence factors yet to be characterized. Several other potential adherence factors have been described for O157:H7 and/or non-O157:H7 strains, although the significance of these factors in human disease is not as well established as intimin. One potential adhesin is a large 362-kDa protein (ToxB) encoded on the 93-kb plasmid that is present in O157:H7 and other EHEC strains 46 . This protein shares sequence similarity with the large Clostridium toxin family, and to the EPEC LifA protein 33 and the Efa-1 protein that has been implicated as an adhesin in non-O157:H7 EHEC strains 34 . This plasmid ( pO157 ) 47 , also encodes an RTX (repeats in toxin) toxin that is similar to the UPEC haemolysin, a serine protease (EspP), a catalase and the StcE protein. StcE cleaves the C1 esterase inhibitor (C1-INH) of the complement pathway and could potentially contribute to the tissue damage, intestinal oedema and thrombotic abnormalities that are seen in EHEC infections 48 . The genome sequence of O157:H7 revealed numerous chromosomal islands (see below) that encode additional potential virulence factors. Included among these potential factors are novel fimbriae, iron uptake and utilization systems 49 , and a urease that is similar to those produced by Klebsiella and other urinary tract pathogens 50 .

Enterotoxigenic E. coli (ETEC). ETEC causes watery diarrhoea, which can range from mild, self-limiting disease to severe purging disease. The organism is an important cause of childhood diarrhoea in the developing world and is the main cause of diarrhoea in travellers to developing countries 2 .

ETEC colonizes the surface of the small bowel mucosa and elaborates enterotoxins, which give rise to intestinal secretion. Colonization is mediated by one or more proteinaceous fimbrial or fibrillar colonization factors (CFs), which are designated by CFA (colonization factor antigen), CS (coli surface antigen) or PCF (putative colonization factor) followed by a number. More than 20 antigenically diverse CFs have been characterized, yet epidemiological studies indicate that approximately 75% of human ETEC express either CFA/I, CFA/II or CFA/IV 51 . Antibodies to CFAs might ameliorate ETEC colonization and disease. ETEC are also an important cause of diarrhoeal disease in animals and these animal strains express fimbrial intestinal colonization factors, such as K88 and K99, which are not found in human ETEC strains.

ETEC enterotoxins belong to one of two groups: the heat-labile enterotoxins (LTs) and the heat-stable enterotoxins (STs). ETEC strains might express only an LT, only an ST, or both LTs and STs. LTs are a class of enterotoxins that are closely related in structure and function to cholera enterotoxin (CT), which is expressed by Vibrio cholerae 52 . The LT that is found predominantly in human isolates (LT-I; a related protein called LT-II is found in some animal ETEC isolates) has ∼ 80% amino acid identity with CT and, like CT, consists of a single A subunit and five identical B subunits. The B subunits mediate binding of the holotoxin to the cell surface gangliosides GM1 and GD1b, and the A subunit is responsible for the enzymatic activity of the toxin. LT has ADP-ribosyl transferase activity and transfers an ADP-ribosyl moiety from NAD to the α-subunit of the stimulatory G protein — a regulatory protein of the basolateral membrane that regulates adenylate cyclase. The resulting permanent activation of adenylate cyclase leads to increased levels of intracellular cAMP, activation of cAMP-dependent kinases and the eventual activation of the main chloride channel of epithelial cells — the cystic fibrosis transmembrane conductance regulator (CTFR). The net result of CFTR phosphorylation is increased Cl − secretion from secretory crypt cells, which leads to diarrhoea (reviewed in Ref. 11 ). LT can also stimulate prostaglandin synthesis and stimulate the enteric nervous system; both of these activities can also lead to stimulation of secretion and inhibition of absorption 11 . LT is also a potent mucosal adjuvant independent of its toxic activity 53 and has been incorporated into numerous vaccine candidates containing a variety of antigens, resulting in increased antibody responses to these antigens when they are delivered orally, nasally or even transdermally.

STs are small, single-peptide toxins that include two unrelated classes — STa and STb — which differ in both structure and mechanism of action. Only toxins of the STa class have been associated with human disease 2 . The mature STa toxin is a ∼ 2-kDa peptide, which contains 18 or 19 amino acid residues, six of which are cysteines that form three intramolecular disulphide bridges (reviewed in Ref. 11 ). The main receptor for STa is a membrane-spanning guanylate cyclase; binding of STa to guanylate cyclase stimulates guanylate cyclase activity, leading to increased intracellular cGMP, which, in turn, activates cGMP-dependent and/or cAMP-dependent kinases and, ultimately, increases secretion. Interestingly, intestinal guanylate cyclase is the receptor for an endogenous ligand called guanylin 54 , which has a similar structure to that of STa. So the ST family seems to represent a case of molecular mimicry. The STb toxin is associated with animal disease and is a 48-amino-acid peptide containing two disulphide bonds (reviewed in Ref. 55 ). STb can elevate cytosolic Ca 2+ concentrations, stimulate the release of prostaglandin E 2 and stimulate the release of serotonin, all of which are mechanisms that could lead to increased ion secretion.

ETEC is largely a pathogen of developing countries, and it is well known that these countries typically have a low rate of colon cancer. Pitari et al . 56 have reported that STa suppresses colon cancer cell proliferation through a guanylyl cyclase C-mediated signalling cascade. So the high prevalence of ETEC in developing countries might have a protective effect against this important disease, and indicates that infectious diseases might exist in a complex evolutionary balance with their human populations.

Enteroaggregative E. coli (EAEC). EAEC are increasingly recognized as a cause of often persistent diarrhoea in children and adults in both developing and developed countries, and have been identified as the cause of several outbreaks worldwide. At present, EAEC are defined as E. coli that do not secrete LT or ST and that adhere to HEp-2 cells in a pattern known as auto-aggregative, in which bacteria adhere to each other in a 'stacked-brick' configuration 2 . It is likely that this definition encompasses both pathogenic and non-pathogenic clones, and it remains controversial as to whether all the EAEC have any common factors that contribute to their shared adherence phenotype. Nevertheless, at least a subset of EAEC are proven human pathogens.

The basic strategy of EAEC infection seems to comprise colonization of the intestinal mucosa, probably predominantly that of the colon, followed by secretion of enterotoxins and cytotoxins 57 . Studies on human intestinal explants indicate that EAEC induces mild, but significant, mucosal damage 58 — these effects are most severe in colonic sections. Mild inflammatory changes are observed in animal models 59 and evidence indicates that at least some EAEC strains might be capable of limited invasion of the mucosal surface 60 , 61 . The most dramatic histopathological finding in infected animal models is the presence of a thick layer of auto-aggregating bacteria adhering loosely to the mucosal surface. EAEC prototype strains adhere to HEp-2 cells and intestinal mucosa by virtue of fimbrial structures known as aggregative adherence fimbriae (AAFs) 62 , 63 , 64 , which are related to the Dr family of adhesins. At least four allelic variants of AAFs exist, but importantly, each is present in only a minority of strains. It should be noted, however, that not all EAEC strains adhere by virtue of AAFs. A recently described protein called dispersin 65 forms a loosely associated layer on the surface of EAEC strains and seems to counter the strong aggregating effects of the AAF adhesin, perhaps facilitating spread across the mucosal surface or penetration of the mucous layer. An additional surface structure that is potentially involved in causing inflammation is a novel EAEC flagellin protein that induces IL-8 release 66 . Release of this cytokine can stimulate neutrophil transmigration across the epithelium, which can itself lead to tissue disruption and fluid secretion.

Several toxins have been described for EAEC. Two such toxins are encoded by the same chromosomal locus on opposite strands. The larger gene encodes an autotransporter protease with mucinase activity called Pic; the opposite strand encodes the oligomeric enterotoxin that is known as Shigella enterotoxin 1 (ShET1), owing to its presence in most strains of Shigella flexneri 2a 67 , 68 . The mode of action of ShET1 is not yet understood, but it might contribute to the secretory diarrhoea that accompanies EAEC and Shigella infection. A second enterotoxin that is present in many EAEC strains is enteroaggregative E. coli ST (EAST1), a 38-amino-acid homologue of the ETEC STa toxin 69 . It is conceivable that EAST1 could contribute to watery diarrhoea in EAST1-positive strains; however, the EAST1 gene ( astA ) can also be found in many commensal E. coli isolates, and therefore the role of EAST1 in diarrhoea remains an open question 70 . Many EAEC strains secrete an autotransporter toxin called Pet, which is encoded on the large virulence plasmid in close proximity to the gene encoding the AAF. Pet has enterotoxic activity and can also potentially lead to cytoskeletal changes and epithelial-cell rounding by cleavage of the cytoskeletal protein spectrin 71 .

Although no single virulence factor has been irrefutably associated with EAEC virulence, epidemiological studies implicate a 'package' of plasmid-borne and chromosomal virulence factors, similar to the virulence factors of other enteric pathogens. Several EAEC virulence factors are regulated by a single transcriptional activator called AggR, which is a member of the AraC family of transcriptional activators 64 (J.P.N., unpublished data). One consistent observation from studies involving EAEC epidemiology is the association of the AggR regulon with diarrhoeal disease. Jiang et al . have recently shown that the presence of genes associated with the AggR regulon is predictive of significantly increased concentrations of faecal IL-8 and IL-1 in patients with diarrhoea caused by EAEC 72 . We suggest that the term 'typical EAEC' should be reserved for strains carrying AggR and at least a subset of AggR-regulated genes (for which the traditional EAEC probe is an adequate marker), and that the term 'atypical EAEC' be used for strains lacking the AggR regulon.

Enteroinvasive E. coli (EIEC). EIEC are biochemically, genetically and pathogenically closely related to Shigella spp. Numerous studies have shown that Shigella and E. coli are taxonomically indistinguishable at the species level 73 , 74 , but, owing to the clinical significance of Shigella , a nomenclature distinction is still maintained. The four Shigella species that are responsible for human disease, S. dysenteriae , S. flexneri , Shigella sonnei and Shigella boydii, cause varying degrees of dysentery, which is characterized by fever, abdominal cramps and diarrhoea containing blood and mucous. EIEC might cause an invasive inflammatory colitis, and occasionally dysentery, but in most cases EIEC elicits watery diarrhoea that is indistinguishable from that due to infection by other E. coli pathogens 2 . EIEC are distinguished from Shigella by a few minor biochemical tests, but these pathotypes share essential virulence factors. EIEC infection is thought to represent an inflammatory colitis, although many patients seem to manifest secretory, small bowel syndrome. The early phase of EIEC/ Shigella pathogenesis comprises epithelial cell penetration, followed by lysis of the endocytic vacuole, intracellular multiplication, directional movement through the cytoplasm and extension into adjacent epithelial cells (reviewed in Ref. 75 ). Movement within the cytoplasm is mediated by nucleation of cellular actin into a 'tail' that extends from one pole of the bacterium. In addition to invasion into and dissemination within epithelial cells, Shigella (and presumably EIEC) also induces apoptosis in infected macrophages 76 . Genes that are required to effect this complex pathogenetic scheme are present on a large virulence plasmid that is found in EIEC and all Shigella species. The sequence of the 213-kb virulence plasmid of S. flexneri (pWR100) indicates that this plasmid is a mosaic that includes genetic elements that were initially carried by four plasmids 77 . One-third of the plasmid is composed of insertion sequence (IS) elements, which are undoubtedly important in the evolution of the virulence plasmid. This plasmid encodes a type III secretion system (see below) and a 120-kDa outer-membrane protein called IcsA, which nucleates actin by the binding of N-WASP 8 , 78 . The growth of actin micofilaments at only one bacterial pole induces movement of the organism through the epithelial cell cytoplasm. This movement culminates in the formation of cellular protrusions that are engulfed by neighbouring cells, after which the process is repeated. Although EIEC are invasive, dissemination of the organism past the submucosa is rare.

Much of EIEC/ Shigella pathogenesis seems to be the result of the multiple effects of its plasmid-borne type III secretion system. This type III secretion system secretes multiple proteins, such as IpaA, IpaB, IpaC and IpgD, which mediate epithelial signalling events, cytoskeletal rearrangements, cellular uptake, lysis of the endocytic vacuole and other actions (reviewed in Refs 79 , 80 ). The type III secretion system apparatus, which is encoded by mxi and spa genes, enables the insertion of a pore containing IpaB and IpaC proteins into host cell membranes. In addition to pore formation, IpaB has several functions, such as binding to the signalling protein CD44, thereby triggering cytoskeletal rearrangements and cell entry, and binding to the macrophage caspase 1, resulting in apoptosis and release of IL-1 from macrophages. IpaC induces actin polymerization, which leads to the formation of cell extensions by activating the GTPases Cdc42 and Rac. The actin polymerization activity resides in the carboxy terminus of IpaC, whereas the amino terminus of this protein is involved in lamellipodial extensions. Conversely, IpaA binds to vinculin and induces actin depolymerization, thereby helping to organize the extensions that are induced by IpaC into a structure that enables bacterial entry. The translocated effector protein IpgD is a potent inositol 4-phosphatase that helps to reorganize host-cell morphology by uncoupling the cellular plasma membrane from the actin cytoskeleton, which leads to membrane blebbing 81 . Although the extensively characterized type III secretion system is essential for the invasiveness characteristic of EIEC and Shigella species, additional virulence factors have been described, including the plasmid-encoded serine protease SepA, the chromosomally encoded aerobactin iron-acquisition system and other secreted proteases that are encoded by genes present on pathogenicity islands (see below).

Diffusely adherent E. coli (DAEC). DAEC are defined by the presence of a characteristic, diffuse pattern of adherence to HEp-2 cell monolayers. DAEC have been implicated as a cause of diarrhoea in several studies, particularly in children >12 months of age 2 , 82 . Approximately 75% of DAEC strains produce a fimbrial adhesin called F1845 or a related adhesin (Ref. 83 ; J.P.N., unpublished observations); F1845 belongs to the Dr family of adhesins, which use DAF, a cell-surface glycosylphosphatidylinositol-anchored protein, which normally protects cells from damage by the complement system, as the receptor 84 , 85 , 86 . DAEC strains induce a cytopathic effect that is characterized by the development of long cellular extensions, which wrap around the adherent bacteria ( Fig. 1 ). This characteristic effect requires binding and clustering of the DAF receptor by Dr fimbriae 85 . All members of the Dr family (including UPEC as well as the DAEC strain C1845) elicit this effect 83 . Binding of Dr adhesins is accompanied by the activation of signal transduction cascades, including activation of PI-3 kinase 86 . Peiffer et al . have reported that infection of an intestinal cell line by strains of DAEC impairs the activities and reduces the abundance of brush-border-associated sucrase-isomaltase and dipeptidylpeptidase IV 87 . This effect is independent of the DAF-associated pathway described above, and therefore provides a feasible mechanism for DAEC-induced enteric disease and also indicates the presence of virulence factors in DAEC other than Dr adhesins. Tieng et al . 7 have proposed that DAEC might induce expression of MICA by intestinal epithelial cells, indicating that DAEC infection could be pro-inflammatory; this effect could potentially be important in the induction of inflammatory bowel diseases.

Uropathogenic E. coli (UPEC). The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common infecting agent at this site. The subset of E. coli that causes uncomplicated cystitis and acute pyelonephritis is distinct from the commensal E. coli strains that comprise most of the E. coli populating the lower colon of humans. E. coli from a small number of O serogroups (six O groups cause 75% of UTIs) have phenotypes that are epidemiologically associated with cystitis and acute pyelonephritis in the normal urinary tract, which include expression of P fimbriae, haemolysin, aerobactin, serum resistance and encapsulation. Clonal groups and epidemic strains that are associated with UTIs have been identified 88 , 89 .

Although many UTI isolates seem to be clonal, there is no single phenotypic profile that causes UTIs. Specific adhesins, including P (Pap), type 1 and other fimbriae (such as F1C, S, M and Dr), seem to aid in colonization 90 , 91 . Several toxins are produced, including haemolysin, cytotoxic necrotizing factor and an autotransported protease known as Sat. These virulence factors are found in differing percentages among various subgroups of UPEC 92 . Uropathogenic strains possess large and small pathogenicity islands containing blocks of genes that are not found in the chromosome of faecal strains. Availability of the genome sequence of E. coli CFT073 (Ref. 93 ) and efforts by other investigators to identify virulence genes by SIGNATURE-TAGGED MUTAGENESIS 94 and other methods have allowed the development of a model of pathogenesis for UPEC ( Fig. 4 ).

The figure shows the different stages of a urinary tract infection. Panels 2, 4, 5 and 11 are courtesy of N. Gunther, A. Jansen, X. Li and D. Auyer (University of Maryland), respectively. CFU, colony-forming units; PMNs, polymorphonuclear leukocytes.

It is likely that infection begins with the colonization of the bowel with a uropathogenic strain in addition to the commensal flora. This strain, by virtue of factors that are encoded in pathogenicity islands, is capable of infecting an immunocompetent host, as it colonizes the periurethral area and ascends the urethra to the bladder ( Fig. 4 ). Between 4 and 24 hours after infection, the new environment in the bladder selects for the expression of type 1 fimbriae 95 , which have an important role early in the development of a UTI 96 . Type 1 fimbriated E. coli attach to mannose moieties of the uroplakin receptors that coat transitional epithelial cells 97 . Attachment triggers apoptosis and exfoliation; for at least one strain, invasion of the bladder epithelium is accompanied with formation of pod-like bulges on the bladder surface that contain bacteria encased in a polysaccharide-rich matrix surrounded by a shell of uroplakin 98 . It is argued that invaded epithelial cells containing a tightly packed bacterial 'biofilm' could act as a reservoir for recurrent infection 97 , 98 , and indeed, in some cases of recurrent infection, the same serotype is encountered. However, a number of studies have identified different serotypes as being responsible for the recurring infection, an observation that is not consistent with this hypothesis. Iron acquisition and the ability to grow in urine are also crucial for survival.

In strains that cause cystitis, type 1 fimbriae are continually expressed and the infection is confined to the bladder 96 . In pyelonephritis strains, the invertible element that controls type 1 fimbriae expression turns to the 'off' position and type 1 fimbriae are less well expressed 95 . It could be argued that this releases the E. coli strain from bladder epithelial cell receptors and allows the organism to ascend through the ureters to the kidneys, where the organism can attach by P fimbriae to digalactoside receptors that are expressed on the kidney epithelium 99 , 100 . At this stage, haemolysin could damage the renal epithelium 101 and, together with other bacterial products including LPS, an acute inflammatory response recruits PMNs to the site. Haemolysin has also been shown to induce Ca 2+ oscillations in renal epithelial cells, resulting in increased production of IL-6 and IL-8 (Ref. 102 ). Secretion of Sat, a vacuolating cytotoxin, damages glomeruli and is cytopathic for the surrounding epithelium 103 . In some cases, the barrier that is provided by the one-cell-thick proximal tubules can be breached and bacteria can penetrate the endothelial cell to enter the bloodstream, leading to bacteraemia.

Meningitis/sepsis-associated E. coli (MNEC). This E. coli pathotype is the most common cause of Gram-negative neonatal meningitis, with a case fatality rate of 15–40% and severe neurological defects in many of the survivors 104 , 105 . The incidence of infants with early-onset sepsis owing to E. coli infection seems to be increasing, while infection by Gram-positive organisms decreases 106 . As with E. coli pathotypes that have a well-defined genetic basis for virulence, strains that cause meningitis are represented by only a limited number of O serogroups, and 80% of the strains are of the K1 capsule type. One interesting difference between MNEC and E. coli that cause intestinal or urinary tract infections is that although the latter strains can be readily transmitted by urine or faeces, infection of the central nervous system offers no obvious advantage for the selection and transmission of virulent MNEC strains.

E. coli that cause meningitis are spread haematogenously. Levels of bacteraemia correlate with the development of meningitis 107 ; for example, bacteraemias of >10 3 colony forming units per ml of blood are significantly more likely to lead to the development of meningitis than in individuals with lower colony forming units per ml in their blood. These bacteria translocate from the blood to the central nervous system without apparent damage to the blood–brain barrier, which indicates a transcytosis process. Electron micrographs imply entry by a zippering mechanism in a process that does not affect transendothelial electrical resistance 108 . This indicates that the host-cell membrane is not significantly disrupted during entry of the bacterium. Two models for studying MNEC have been developed: a monolayer of brain microvascular endothelial cells 109 and an intact animal model using 5-day-old rats 110 .

As for other E. coli pathotypes, the genomes of these extraintestinal K1 strains have additional genes that are not found in the commensal E. coli K-12 strains. In genomic comparisons, the genome of E. coli RS218, a meningitis-associated strain, was found to have at least 500 kb of additional genes inserted in at least 12 loci compared with E. coli K-12 (Refs 111 , 112 ). In addition, strain RS218 harbours a 100-kb plasmid, on which at least one virulence factor has been localized 113 .

Some insights into the mechanism of pathogenesis of these strains have been obtained. K1 strains use S fimbriae to bind to the lumenal surfaces of brain microvascular endothelium in neonatal rats 114 . Invasion requires the outer-membrane protein OmpA to bind to the GlcNAcβ1-4GlcNAc epitope of the brain microvascular endothelial cell receptor glycoprotein 115 . Other membrane proteins — for example, IbeA, IbeB, IbeC and AslA — are also required for invasion (reviewed in Ref. 116 ). Invasion correlates with microaerobic growth and iron supplementation 117 . CNF1 is required for invasion 113 , as is the K1 capsule, which elicits serum resistance and has antiphagocytic properties. In an experimental model, strains that express K1 capsule proteins and those that do not were able to cross the blood–brain barrier, but only the K1-expressing strains survived 118 . As a consequence of invasion, actin cytoskeletal rearrangement occurs and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin is induced 119 . In addition, a substantial list of in vivo -induced genes, including those that encode iron-acquisition systems, was compiled using in vivo expression technology ( IVET ) in conjunction with a murine model of septicaemic infection 120 .

Other potential E. coli pathotypes. Several other potential E. coli pathotypes have been described, but none of these are as well established as the pathotypes described above ( Box 1 ). Among the most intriguing of these potential pathogens are strains of E. coli that are associated with Crohn's Disease, which are known as adherent-invasive E. coli (AIEC) 121 . No unique genetic sequences have yet been described for AIEC strains, but such strains can invade and replicate within macrophages without inducing host-cell death and can induce the release of high amounts of tumour-necrosis factor (TNF)-α, a characteristic which could lead to the intestinal inflammation that is characteristic of Crohn's Disease. An inflammatory process, together with necrosis of the intestinal epithelium, are characteristics of necrotizing enterocolitis (NEC), an important cause of mortality and long-term morbidity in pre-term infants. The ability of some E. coli strains to transcytose through epithelial cell monolayers has been hypothesized to contribute to NEC 122 . Necrotoxic E. coli (NTEC) produce either CNF1 or CNF2 and have been associated with disease in both humans and animals 123 . Strains that are known as cell-detaching E. coli (CDEC) have been isolated from children with diarrhoea and the characteristic ability of these strains to detach cultured epithelial cells from glass or plastic has been associated with the production of haemolysin 124 . The relationships among the NEC-associated strains, NTEC and CDEC, have not yet been clearly established. The genes encoding CDT are infrequently present in E. coli strains and no significant association with disease has yet been found for this toxin. CDT is usually found in strains that possess other virulence factors, such as CNF, Stx and the LEE. However, recent information indicates that CDT can be encoded by four distinct genetic variants in E. coli and so earlier epidemiological studies using only one or two cdt genes as probes should be re-evaluated 125 . In at least one strain, the cdt genes are contained on a bacteriophage 126 , which could account for the presence of this toxin in a number of different E. coli pathotypes.

A poorly characterized subset of E. coli infections outside the gastrointestinal or urinary tract is a group implicated in intra-abdominal infections (IAIs), including abscesses, wounds, appendicitis and peritonitis. The initial microflora at the site of an IAI is polymicrobial, but E. coli and the strictly anaerobic Bacteroides fragilis are often isolated from these abscesses. A recent study indicates that a novel haem-binding protein, known as the 'haemoglobin-binding protease' (Hbp), is significantly associated with E. coli strains isolated from IAIs compared with those E. coli strains isolated from blood, urine or faeces 127 . Purified Hbp was shown to be capable of delivering haem to B. fragilis , indicating a synergy in abscess formation whereby E. coli provides iron from haem to B. fragilis to overcome iron restrictions imposed by the host. Interestingly, Hbp is identical to Tsh, which is an autotransporter haemagglutinin that is associated with APEC, thereby indicating that this protein can contribute to at least two different infectious diseases — IAIs in humans and respiratory tract infections in poultry 127 .

Mobile genetic elements. A striking feature of pathogenic E. coli is the association of genes that encode virulence factors with mobile genetic elements ( Fig. 5 ). This was first shown more than 30 years ago with ETEC strains, in which enterotoxic activity was transferred together with a self-transmissible plasmid. In many cases, these 'Ent' plasmids were also shown to encode antibiotic resistance. There are now numerous examples of plasmids that encode crucial virulence factors of pathogenic E. coli , including plasmids in EAEC that encode fimbriae and toxins, plasmids in EIEC/ Shigella that encode a type III secretion system and invasion factors, the EPEC EAF plasmid, which encodes BFP, and the pO157 plasmid of EHEC, which encodes accessory toxins. Although many of these plasmids are self-transmissible, some lack conjugation genes and can only be transferred with a conjugative plasmid. For ETEC, the genes that encode both LT and ST are found on plasmids, but some estA genes encoding STa are on transposons that can be inserted into either plasmids or the chromosome. One IS element has been described that contains the astA gene encoding the EAST1 toxin, completely embedded in a large putative transposase gene, the coding sequence of which is on the same strand but in the −1 reading frame relative to astA 128 .

E. coli virulence factors can be encoded by several mobile genetic elements, including transposons (Tn) (for example, heat stable enterotoxin (ST) of ETEC), plasmids (for example, heat-labile enterotoxin (LT) of ETEC and invasion factors of EIEC), bacteriophage (for example, Shiga toxin of EHEC) and pathogenicity islands (PAIs) — for example, the locus of enterocyte effacement (LEE) of EPEC/EHEC and PAIs I and II of UPEC. Commensal E. coli can also undergo deletions resulting in 'black holes', point mutations or other DNA rearrangements that can contribute to virulence. These additions, deletions and other genetic changes can give rise to pathogenic E. coli forms capable of causing diarrhoea (EPEC, EHEC, EAEC DAEC), dysentery (EIEC), haemolytic uremic syndrome (EHEC), urinary tract infections (UPEC) and meningitis (MNEC). HUS, haemolytic uremic syndrome; UTI, urinary tract infection.

The main virulence factor of EHEC, Stx, is encoded on a lambda-like bacteriophage; acquisition of this phage was a key step in the evolution of EHEC from EPEC 45 . The EHEC EDL933 genome sequence contains 18 regions with homology to known bacteriophages, but most seem to be incomplete phage genomes 49 . Although only the Stx phage seems to be capable of lytic growth and production of infectious particles, these cryptic phage sequences enable the continued evolution of these strains by homologous recombination of phages into different chromosomal sites. The ability to produce Stx can be readily transmitted by transduction of the genes encoding Stx phage to K-12 or commensal E. coli , but this step is probably insufficient to confer virulence because non-O157:H7 E. coli strains containing stx genes without other EHEC virulence factor genes can be readily isolated from commercial meat products. This observation reinforces the concept that a single gene is insufficient to convert commensal E. coli to pathogenic E. coli , and that instead a combination of genes encoding toxins, colonization factors and other functions are required to make E. coli pathogenic.

PAIs are large genomic regions (10–200 kb) that are present in the genomes of pathogenic strains but absent from the genomes of non-pathogenic members of the same or related species (reviewed in Ref. 129 ). PAIs are typically associated with tRNA genes, have a different G+C content compared with the host DNA and often carry cryptic or functional genes that encode mobility factors, such as integrases, transposases and IS elements. PAIs were first described in pathogenic E. coli and have subsequently been described in several Gram-negative and Gram-positive bacteria. The first PAIs were described in UPEC strain 536, which contains at least four such islands 130 . The PAI II 536 island is 100 kb in size, is inserted at the leuX tRNA gene at minute 97 on the E. coli chromosome and encodes haemolysin and P fimbriae. This island is flanked by 18-bp direct repeats, which facilitate deletion of the entire island at a relatively high frequency.

The first PAI to be described in diarrhoeagenic E. coli was the LEE PAI in EPEC and EHEC 21 . As described above, the LEE encodes a type III secretion system and other factors that are responsible for the A/E histopathology. In EPEC strain E2348/69 and EHEC strain O157:H7, the LEE is inserted at the selC tRNA gene, which is also the site of insertion of the PAI I 536 island of UPEC. The insertion of two different PAIs at the same chromosomal site in EPEC/EHEC and UPEC indicates the presence of 'hot spots' in the E. coli chromosome into which different PAIs can insert and give rise to different E. coli pathotypes. The 35-kb LEE from E2348/69 contains 41 open reading frames that are highly conserved among EPEC and EHEC strains, as well as rabbit and other animal strains of EPEC that produce A/E lesions. In some E. coli strains, the LEE PAI is immediately adjacent to genes that encode other potential virulence factors, such as the efa1/lifA gene, to form a larger PAI of 59.5 kb 131 . The LEE of one rabbit strain is contained on a ∼ 85-kb PAI that contains an intact integrase gene and is flanked by direct repeats. This PAI is capable of spontaneous deletion and site-specific integration into the pheU tRNA locus of K-12 (Ref. 131 ). The prototypic LEE of E2348/69 contains no direct repeats or mobility genes and seems to be incapable of spontaneous deletion or transfer, which indicates that this PAI has evolved to the point that it has lost the genetic elements that were responsible for the initial integration into the chromosome.

PAIs have also been described for EAEC, EIEC/ Shigella , MNEC and some ETEC strains (reviewed in Refs 132 – 134 ). Some PAIs are unique to individual pathotypes, whereas other PAIs are found in multiple pathotypes. The she (Shi-I) PAI is present in EAEC, where it encodes the ShET1 enterotoxin and the autotransporter toxin Pic. The high pathogenicity island (HPI) was originally described in Yersinia , but is also present in most strains of EAEC, DAEC and UPEC, and in some strains of EIEC, ETEC, EPEC and EHEC, as well as some Klebsiella and Citrobacter strains 135 . The HPI contains genes that are involved in regulation, biosynthesis and uptake of the siderophore yersiniabactin.

The inverse of PAIs are 'black holes', which refers to the deletion of blocks of genes in commensal or K-12 E. coli that lead to increased virulence. In EIEC/ Shigella , lack of the cadA gene, which encodes lysine decarboxylase (LDC) in K-12, enables activity of an enterotoxin which is normally inhibited by the product of the LDC reaction — cadaverine 136 . In many EIEC strains, the cadC gene that encodes a regulator of cadA is preferentially mutated, which results in the same phenotype 137 . EIEC/ Shigella also have a large number of pseudogenes (see below), which might also comprise functional 'black holes'. Although the genes encoding E. coli virulence factors are usually either present or absent, single-nucleotide polymorphisms (SNPs) that contribute to virulence have been found in the genes that encode the FimH and Dr adhesins 138 .

Genomic sequences. Prior to the determination of the complete genomic sequence for a pathogenic strain of E. coli it was anticipated that these pathogens differed from K-12 primarily by the presence of a limited number of PAIs, plasmids and phage that encoded specific virulence factors. However, when the first pathotype was sequenced — namely two different strains of EHEC O157:H7 — the extent of lateral gene transfer was found to be far greater than had been anticipated. EHEC strain EDL933 contains nearly 1,400 novel genes scattered throughout 177 discrete regions of DNA greater than 50 bp in size called O-islands; these regions total 1.34 Mb of DNA that is not present in K-12 (Ref. 49 ). Almost as surprising was the fact that although the two strains shared a 4.1-Mb 'backbone' of common sequences, EDL933 lacked 0.53 Mb of DNA that was present in K-12 in 234 'K-islands' (>50 bp). The absence of a substantial amount of K-12 DNA in other E. coli pathotypes was shown in a recent DNA array study in which up to 10% of E. coli K-12 open reading frames were not detected in several pathogenic and non-pathogenic E. coli strains 139 .

The striking mosaic structure of EHEC was further shown by the determination of the UPEC genome sequence, which at 5.2 Mb is similar in size to that of EHEC 93 . UPEC strain CFT073 contains 2,004 genes in 247 islands that are not present in K-12. In contrast to the striking conservation of the core LEE PAI in EPEC and EHEC, substantial differences were seen between the large PAIs of CFT073 and two other well-studied UPEC strains — J96 and 536. The analyses indicated that extraintestinal pathogenic E. coli strains arose independently from multiple clonal lineages. Interestingly, when the predicted proteins from all three strains, K-12, EHEC and UPEC, were compared, only 39.2% of the combined (nonredundant) set of proteins are common to all three strains 93 .

As noted above, several studies using DNA hybridization, multilocus enzyme electrophoresis and sequencing of a small number of genes indicates that Shigella species clearly fall taxonomically within the E. coli species 74 . The genome sequence of S. flexneri 2a further supports this grouping and exhibits the backbone and island mosaic structure of the genomes of the E. coli pathogens 73 . The 4.599-Mb genome size is closer to that of K-12 (4.639 Mb) than to EHEC and UPEC, and the 70.6% of K-12 genes that are found in S. flexneri is of a similar magnitude to the 74.3% of K-12 genes that are found in UPEC CFT073. However, UPEC contains an additional 1,827 proteins that are not found in K-12, whereas S. flexneri contains only 205 proteins that are not found in K-12, thereby indicating that S. flexneri is more similar to K-12 than is UPEC CFT073. The S. flexneri genome is notable for its large number of IS elements — which constitute 6.7% (309.4 kb) of the chromosome — and for the large number (372) of pseudogenes present — which constitute 8.1% of the genome. These pseudogenes arose by several mechanisms, including single-nucleotide insertions or deletions, point mutations and IS-element insertions. Interestingly, phenotypic tests that have traditionally been used to distinguish E. coli from S. flexneri , such as lack of motility, utilization of various carbon sources and the requirement for NAD, are largely the result of pseudogenes. Whether these pseudogenes are advantageous, disadvantageous, or neutral cannot be stated at this time.

Regulation. Consistent with the fact that E. coli virulence factors are typically encoded on 'foreign' DNA that is not contained in commensal E. coli strains, the expression of many virulence factor genes is frequently regulated by transcriptional regulators that are also encoded on pathogenicity islands or plasmids. One such pathogen-specific regulator is the LEE-encoded Ler protein, which positively regulates the EPEC/EHEC genes encoding the type III secretion system that are also found on the LEE 140 . Another example is the PapB regulator of the pap operon encoded on PAIs in UPEC 141 . In some instances, a plasmid-encoded regulator can activate transcription of chromosomal genes — for example, regulators such as the regulatory cascade formed by the EPEC plasmid-encoded regulator (Per) that regulates the LEE-encoded regulator, Ler ( Fig. 6 ). Many pathogen-specific regulators belong to the AraC family of transcriptional activators, such as Per (EPEC), AggR (EAEC), VirF (EIEC) and Rns (ETEC).

The attaching and effacing histopathology induced by EPEC and EHEC is encoded by the locus of enterocyte effacement (LEE) pathogenicity island, which contains five major polycistronic operons designated LEE1–5 . Expression of the LEE genes is regulated by EPEC-specific regulators (depicted in green) and generic E. coli regulators (depicted in yellow). The first open reading frame of the LEE1 operon encodes the LEE-encoded regulator, Ler, which positively regulates expression of other LEE operons by counteracting the repressive effects of H-NS 140 , 148 . Ler also regulates expression of the EspC enterotoxin that is produced by many EPEC strains and potentially other virulence factors. Expression of Ler is itself regulated by several factors, including IHF 149 , FIS 150 and BipA 151 , and quorum sensing through the QseA regulator 152 . Quorum sensing also regulates other factors that are potentially involved in virulence, such as flagella, through the QseBC two-component regulator 153 . In EPEC, but not EHEC, expression of Ler is positively regulated by the products of the per (plasmid-encoded regulator) 154 locus, which consists of three open reading frames, perA , perB and perC ; PerA (BfpT) also regulates the bfp genes encoding a type IV pilus 155 . In acidic conditions, the per genes are repressed by GadX, which activates the gadAB genes involved in acid resistance 156 . This dual action of GadX could prevent premature expression of virulence factors in the stomach while enhancing survival of the organism until it reaches more alkaline conditions in the small intestine where expression of virulence factors is induced. Bip, Ig heavy chain binding protein; FIS, factor for inversion stimulation; IHF, integration host factor.

Expression of E. coli virulence factors is not solely regulated by pathogen-specific regulators. A common theme among the various E. coli pathotypes is the exploitation of regulators present in commensal E. coli for the regulation of virulence factor genes that are present only in pathogenic E. coli . For example, the stx 1 gene encoding Shiga toxin is transcribed from the P R ′ promoter that also controls expression of late lambda phage lysis genes, thereby linking toxin expression with a lytic function, which allows release of the toxin 142 . This linkage leads to induction of transcription of both toxin genes and lysis genes by certain antibiotics, causing increased toxin production, increased release of toxin by lysis and increased death in a mouse model 143 . Another example is the EPEC Ler, which in addition to being regulated by Per is also regulated by integrative host factor (IHF), factor for inversion stimulation (FIS) and Ig heavy chain binding protein (BipA) — global regulators of housekeeping genes in K-12 ( Fig. 6 ). Another regulatory system present in K-12 that regulates expression of both housekeeping and virulence factor genes is the AI-2/ luxS quorum sensing (QS) system. QS is a method of intercellular communication that allows unicellular organisms such as E. coli to behave as multi-cellular organisms. A small autoinducer (AI) molecule is produced by many organisms, including E. coli ; AIs can activate the expression of a subset of genes when the microbial population, and therefore the AI concentration, reaches a crucial level. QS regulates the expression of the EPEC and EHEC LEE operons by Ler as well as flagella expression 144 . As the infectious dose of EHEC (10–100 organisms) is too low to make use of QS, a model has been proposed in which EHEC detect the AI signals that are produced by the large concentration of commensal E. coli and other bacteria present in the large intestine 144 . In response to this signal, expression of key virulence factors, including the LEE and Stx, is induced, thereby initiating the disease process. This regulatory mechanism can also be activated by mammalian hormones, such as adrenaline and noradrenaline, in an example of regulatory 'cross-talk' between eukaryotic and prokaryotic organisms 145 .

Regulation of virulence factor expression by physical DNA rearrangements is uncommon in pathogenic E. coli but phase variation is seen with type 1 fimbriae. Transcription of the fim operon that encodes type 1 fimbriae is primarily under the control of an invertible element that contains the promoter responsible for transcription of the main structural subunit. Individual bacterial cells either express the fimbriae over their entire surface or do not express any fimbriae. This phase variation of type 1 fimbriae is controlled at the transcriptional level by the invertible element, which is regulated by the FimB and FimE recombinases 146 . The inversion seems to be regulated during the course of infection, and the orientation of the element correlates with whether UPEC strains remain localized to the bladder. In cystitis infections most of the strains have the invertible element in the 'on' position and express type 1 fimbriae, whereas when they leave the bladder and ascend to the kidneys to cause pyelonephritis, most of the strains have the element in the 'off' position and do not express type 1 fimbriae 95 . The regulation of type 1 fimbriae in UPEC is further complicated by cross-talk between two different adhesion operons, whereby PapB, a key regulator of the pap operon, inhibits type 1 phase variation 141 .

Conclusions

The evolution of pathogenic E. coli that has resulted in formation of distinct pathotypes capable of colonizing the gastrointestinal tract, urinary tract or meninges illustrates how key genetic elements can adapt a strain to distinct host environments. Using E. coli K-12 as a 'base-model', several features can be added (PAIs, plasmids, transposons or phage) or subtracted (black holes or pseudogenes) to modify the base model to adapt to specific environments and to enable these modified strains to cause disease in an immunocompetent human or animal host. This genomic plasticity complicates efforts to categorize the various clusters of pathogenic E. coli strains into sharply delineated pathotypes. The evolutionary process, clearly ongoing, has resulted in a highly versatile species that is capable of colonizing, multiplying in and damaging diverse environments. The host cell activities that are affected by these pathogenic strains of E. coli encompass a broad spectrum of functions, including signal transduction, protein synthesis, mitochondrial function, cytoskeletal function, cell division, ion secretion, transcription and apoptosis. The ability of various E. coli virulence factors to affect such a wide range of cellular functions has led to the use of the various toxins, effectors and cell surface structures as tools to better understand these fundamental eukaryotic processes. Our increased understanding of the mechanisms by which E. coli can cause disease has dramatically changed our perspective of this species that was once dismissed as a harmless commensal of the intestinal tract.

Box 1 | Questions for future research

What are the best methods for the diagnosis of intestinal E. coli pathogens so they can be routinely diagnosed in clinical laboratories and their true significance determined?

What are the factors that allow commensal E. coli strains to colonize the intestine and survive so successfully in this niche?

What is the role of E. coli in Crohn's Disease and possibly other intestinal diseases that were previously considered to be non-infectious in origin?

What is the best way to treat and/or prevent enterohaemorrhagic E. coli infection to prevent the most serious outcome — haemolytic uremic syndrome.

What are the pathogenetic mechanisms and roles of EAEC and DAEC in enteric disease?

What other pathotypes of E. coli are yet to be discovered or yet to evolve?

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Acknowledgements

Work in the authors' laboratories is supported by the National Institutes of Health. We thank J. Girón for providing electron micrographs. We apologize to the numerous investigators whose papers could not be cited due to space constraints.

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James B. Kaper & James P. Nataro

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James B. Kaper & Harry L. T. Mobley

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Research Article

Prevalence and characterization of Escherichia coli isolated from the Upper Oconee Watershed in Northeast Georgia

Roles Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing

Affiliation Department of Microbiology, University of Georgia, Athens, Georgia, United States of America

Roles Investigation, Methodology, Project administration, Supervision, Writing – review & editing

Affiliation Bacterial Epidemiology and Antimicrobial Resistance Research Unit, United States Department of Agriculture, Agricultural Research Service, Athens, Georgia, United States of America

Roles Investigation, Writing – review & editing

Roles Investigation, Methodology, Writing – review & editing

Roles Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing

¶ ‡ CRJ and JGF are joint senior authors on this work.

Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing

* E-mail: [email protected]

• Sohyun Cho, 
• Lari M. Hiott, 
• John B. Barrett, 
• Elizabeth A. McMillan, 
• Sandra L. House, 
• Shaheen B. Humayoun, 
• Eric S. Adams, 
• Charlene R. Jackson, 
• Jonathan G. Frye

• Published: May 8, 2018
• https://doi.org/10.1371/journal.pone.0197005
• Reader Comments

Surface waters are important sources of water for drinking, industrial, agricultural, and recreational uses; hence, contamination of water by fecal, pathogenic, or antimicrobial resistant (AR) bacteria is a major environmental and public health concern. However, very little data is available on prevalence of these bacteria in surface water throughout a watershed. This study aimed to characterize Escherichia coli present in the Upper Oconee Watershed, a mixed-use watershed in Athens, GA, USA for potential pathogenicity and AR. E . coli were enumerated by colony counts, cultured by enrichment and direct plating, and characterized by phylo-groups, diarrheagenic pathotypes, and antimicrobial susceptibility. From the analysis, 99.3% (455/458) of the total samples were positive for E . coli resulting in 496 isolates. E . coli counts were as high as 1.2×10 4 CFU/100 ml, which is above the United States Environmental Protection Agency (U.S. EPA) threshold for recreational water (235 CFU/100 ml based on a one-time measurement). Phylo-groups B2 (31.7%; 157/496) and B1 (30.8%; 153/496) were the most prevalent among the isolates. Enteropathogenic E . coli (EPEC) (19/496) and Shiga toxin-producing E . coli (STEC) (1/496) were the only diarrheagenic pathotypes detected. AR was observed in 6.9% (34/496) of the isolates, 15 of which were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). Tetracycline resistance was most often detected (76.5%; 26/34), followed by ampicillin (32.4%; 11/34), streptomycin (23.5%; 8/34), sulfisoxazole (23.5%; 8/34), and nalidixic acid (14.7%; 5/34). Results from this study showed that E . coli is prevalent in high levels in the Upper Oconee Watershed, suggesting possible widespread fecal contamination. The presence of pathogenic, AR E . coli in the watershed indicates that environmental water can serve as a reservoir of resistant bacteria that may be transferred to humans through drinking and recreational activities.

Citation: Cho S, Hiott LM, Barrett JB, McMillan EA, House SL, Humayoun SB, et al. (2018) Prevalence and characterization of Escherichia coli isolated from the Upper Oconee Watershed in Northeast Georgia. PLoS ONE 13(5): e0197005. https://doi.org/10.1371/journal.pone.0197005

Editor: A. Mark Ibekwe, USDA-ARS Salinity Laboratory, UNITED STATES

Received: February 14, 2018; Accepted: April 24, 2018; Published: May 8, 2018

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: Funded by United States Department of Agriculture, Agricultural Research Service Intramural funding Project Number: 6040-32000-009-00 https://iapreview.ars.usda.gov/research/projects/projects.htm?ACCN_NO=430182 . Centers for Disease Control and Prevention. Broad Agency Announcement to address antibiotic resistance, Agricultural Research Service Sub-Project Number: 6040-32000-009-08R https://www.cdc.gov/drugresistance/solutions-initiative/innovations-to-slow-AR.html .

Competing interests: The authors have declared that no competing interests exist.

Introduction

Escherichia coli , which normally resides in the intestinal flora of warm-blooded animals, including humans, is ubiquitous in the environment and has been used as an indicator of fecal contamination to assess the safety and quality of water [ 1 ]. Although most E . coli strains are harmless, certain strains are pathogenic and cause diseases such as watery diarrhea, bloody diarrhea, urinary tract infection, meningitis, and sepsis, which can lead to death [ 2 , 3 ]. The normally zoonotic bacterial pathogen has been responsible for waterborne outbreaks in humans through contaminated drinking and recreational water not only in developing countries, but also in industrialized countries [ 4 – 9 ]. Environmental water sources are prone to bacterial pollution from both humans and animals. Possible human sources include discharge of wastewater, sewage leaks, and failing septic tanks, as well as municipal, residential, medical, and industrial waste facilities. Animal sources include runoffs from animal farms, land application of animal manure, pet wastes from parks, and wildlife such as raccoons and deer. Since surface waters are often used for recreational and drinking purposes, the presence of pathogenic E . coli in waterways may increase the likelihood of human infections after exposure to these water sources.

The Upper Oconee Watershed, located in the Southern Piedmont of Georgia, USA, is a historically agricultural region that has experienced rapid urban development. While two-thirds of the watershed still remains undeveloped with rural residential, forest and agricultural lands, the remaining land areas have transitioned to urban and suburban residential areas [ 10 ]. The Upper Oconee Watershed is not only impacted by dense residential development, industrialization, and sporadic sewer spills, but also includes land areas heavily devoted to agriculture, including poultry, dairy cattle, and beef cattle production [ 11 , 12 ]. Since the watershed provides water for municipal and recreational purposes, monitoring the water quality is a public health concern. The Upper Oconee Watershed Network (UOWN) is a nonprofit organization dedicated to protecting streams and rivers within the Upper Oconee Watershed [ 13 ]. Since January 2000, the UOWN has been monitoring the surface water and its reporting indicates recurrent fecal contamination of the surface water within the watershed as evidenced by high fecal coliform and E . coli levels [ 11 , 14 – 16 ].

The goal of this study was to investigate seasonal and spatial prevalence and characteristics of E . coli present in the Upper Oconee Watershed in and around Athens, Georgia. Since previous reports only included data on fecal indicator levels within the surface water of the Upper Oconee Watershed [ 11 , 14 – 16 ], the present study attempted to further examine surface water quality for recreational and drinking purposes by investigating each E . coli isolate for its potential to cause disease and its antimicrobial resistance (AR). Environmental water samples were collected each season for two years and fecal contamination was determined by enumerating E . coli colony counts. E . coli was isolated from each water sample and characterized for phylo-group, pathotype, and AR phenotype. Susceptibility testing with 14 antimicrobials that are largely used for treating human and animal infections was used to determine AR phenotypes, because AR E . coli could be a public health concern as they can potentially restrict treatment options in the event of an infection. This study provides unique data on E . coli prevalence and characteristics in a mixed-use watershed that is representative of what residents of rural, urban, and suburban areas may be exposed to through the recreational, agricultural, and municipal use of surface water. Although there have been several studies on E . coli in surface water, most of these studies focused on a single waterway rather than a watershed, and the few studies of watersheds were usually limited in sampling events, sites, or time period. This study is unique in that it provides data on an entire watershed sampled over two years and numerous sampling sites.

Materials and methods

Sampling area.

The rivers and streams sampled in this study were located in the Upper Oconee Watershed (USGS Cataloging unit 03070101). As previously described [ 17 ], the study area is approximately 600 km 2 and located within the lower Appalachian Piedmont of Northeastern Georgia, USA. Sampling sites were located along the North Oconee River (NORO), Middle Oconee River (MIDO), and their tributaries. A part of the study area is developed and densely populated consisting of urban residential areas that depend primarily on community sewers for effluent wastewater [ 17 ]. Other parts of the area mainly consist of forested, agricultural, and rural or suburban residential areas with poultry, dairy, and beef farming, largely depending on private septic systems for effluents [ 17 ]. Sampling sites were selected by the UOWN to represent a range of land uses ( Fig 1 ). Maps and site descriptions for the sampling sites are available from the UOWN website [ 13 ] and in Fig 1 . The exact locations of the sites with the GPS coordinates are in S1 Table . No specific permissions were required to collect water samples from these public access sites and no wildlife, endangered, or protected species were involved in this study.

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• TIFF original image

The map of the Upper Oconee Watershed in Georgia with the enlarged map of the study area. Sampling sites where AR E . coli were isolated are in red, where EPEC were isolated are in blue, where STEC was isolated is in green, and where both AR E . coli and EPEC were isolated are in purple (sites labeled). Other sites, where all E . coli isolates were pan-susceptible and non-pathogenic, are in yellow.

https://doi.org/10.1371/journal.pone.0197005.g001

Water collection and enumeration of E . coli in water samples

One-liter water samples were collected once each season for two years from 2015 Winter to 2016 Fall at different locations in the Upper Oconee Watershed with the assistance of the UOWN volunteers. The number of water samples collected each time varied from 30 to 100, depending on available manpower and access to the sampling sites. Samples were stored at 4°C until processing the next day.

E . coli counts were enumerated in duplicates using Petrifilm ™ E . coli /Coliform count plates (3M ™ , St. Paul, MN, USA) according to manufacturer’s directions. E . coli enumeration was carried out before the filtration of water samples by inoculating two Petrifilm ™ plates with 1 ml of water each per sample. Plates were incubated at 37°C, and colonies were enumerated after 18–20 h of incubation. E . coli counts were averaged from the duplicate plates and expressed as CFU/100 ml.

Isolation and identification of E . coli

Filtration of water samples was performed as previously described [ 17 ]. Briefly, 0.5 g of cellulose filter powder (Aqua Dew ™ , Lahore, Pakistan) was added to water samples, and the water samples were filtered onto 47-mm glass fiber filters of 0.3 μm pore size (Pall Corporation, Ann Arbor, MI, USA), which had been preloaded with another 0.5 g of cellulose filter powder suspended in 15 ml of sterile water. The filter, along with the filter powder, was incubated in 25 ml of 1X buffered peptone water (BD Difco ™ , Franklin Lakes, NJ, USA) for non-selective pre-enrichment of samples. All overnight samples were incubated at 37°C for 18–20 h.

For E . coli isolation, 0.1 ml of each peptone broth enrichment was streaked on a CHROMagar ECC agar plate (CHROMagar Microbiology, Paris, France). One year of samples were also plated onto CHROMagar O157 agar plates (CHROMagar Microbiology). However, this medium yielded no O157 isolates and its use was discontinued (data not shown). In addition to CHROMagar ECC agar plates, m-TEC agar plates (HiCrome ™ , Mumbai, India), which are normally used in the EPA method, were also used for isolation of E . coli during the 2015 Winter and Summer seasons. However, the m-TEC agar did not give consistent results and often failed to yield E . coli isolates, therefore that method was also discontinued. After a 37°C overnight incubation, one colony having the typical appearance of E . coli was selected from each positive plate. Presumptive positive E . coli isolates were then confirmed using the VITEK ® 2 System and the VITEK 2 GN colorimetric identification cards (BioMérieux, Durham, NC, USA) according to manufacturer’s directions. All bacterial isolates were stored in LB (Luria-Bertani) broth (BD Difco ™ ), containing 30% glycerol at -80°C.

Phylogenetic analysis and identification of diarrheagenic pathotypes of E . coli

For genotypic profiling of E . coli , phylo-group and diarrheagenic pathotype identifications were performed. E . coli were grouped into eight phylo-groups, A, B1, B2, C, D, E, F, and cryptic clade I, using the quadruplex phylo-typing PCR method as previously described [ 18 ]. E . coli ATCC 25922 and ATCC BAA-196 were used as control strains. The diarrheagenic pathotypes were determined using PCR methods for detection of the following genes: pCVD, ipaH , est , elt , stx1 , stx2 , eaeA , and hlyA . Using previously described methods, E . coli isolates were characterized as enteroaggregative E . coli (EAEC) (pCVD+) [ 19 ], enteroinvasive E . coli (EIEC) ( ipaH +) [ 20 ], enterotoxigenic E . coli (ETEC) ( est +, elt +) [ 20 ], enterohemorrhagic E . coli (EHEC) ( stx1 + and/or stx2 +, eaeA +, hlyA +) [ 19 ], enteropathogenic E . coli (EPEC) ( eaeA +, stx1 -, stx2 -, hlyA -) [ 21 ], and Shiga toxin-producing E . coli (STEC) ( stx1 + and/or stx2 +, eaeA -, hlyA -) [ 21 ]. The following E . coli strains were included as positive control strains: ATCC 29552 (EAEC), ATCC 35401 (ETEC), ATCC 43893 (EIEC), and ATCC 43895 (EPEC, EHEC, and STEC). The STEC isolate was tested for serogroups O111 and O157 using a method previously described [ 21 ]. PCR was performed as described in the given references using whole-cell templates that were prepared by suspending a single bacterial colony in 200 μl of sterile deionized water. Amplified PCR products were then analyzed by electrophoresis on 2% agarose gel and visualized by staining with ethidium bromide.

Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MIC) of all E . coli isolates were determined by broth-microdilution using the Sensititre ™ semi-automated antimicrobial susceptibility system (TREK Diagnostic Systems Inc., Cleveland, OH, USA) and the Sensititre ™ custom National Antimicrobial Resistance Monitoring System (NARMS) plate CMV3AGNF according to manufacturer’s directions. MICs of the isolates for the 14 antimicrobials were determined, and each isolate was classified as resistant, intermediate, or susceptible to the antimicrobials tested using the breakpoints set by Clinical and Laboratory Standards Institute (CLSI) [ 22 ]. For azithromycin, without CLSI approved breakpoints, the epidemiological cutoff value for wild-type Salmonella (MIC > 16 μg/ml) was used [ 23 , 24 ]. The 14 antimicrobials and the breakpoints (μg/ml) for determining resistances were as follows: amoxicillin/clavulanic acid (≥ 32/16), ampicillin (≥ 32), azithromycin (> 16), cefoxitin (≥ 32), ceftiofur (≥ 8), ceftriaxone (≥ 4), chloramphenicol (≥ 32), ciprofloxacin (≥ 1), gentamicin (≥ 16), nalidixic acid (≥ 32), streptomycin (≥ 64), sulfisoxazole (≥ 512), tetracycline (≥ 16), and trimethoprim/sulfamethoxazole (≥ 4/76). For the analysis, isolates identified as intermediate were considered susceptible to the drug. E . coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213 were used as control strains for MIC determination.

Prevalence of E . coli

A total of 458 water samples were collected from eight seasonal sampling events. The sampling site locations are shown on the map in Fig 1 and listed in S1 Table with each site’s GPS coordinates. The number of sampling sites positive for E . coli and the number of isolates recovered from the sites are shown in Table 1 . E . coli was recovered from 99.3% (455/458) of the total sampling sites, with the recovery rate for each sampling ranging from 96.7% to 100.0%, and a total of 496 E . coli were isolated. Although only one colony was selected from each positive plate, higher number of isolates than the number of sites is due to the use of mTEC agar in addition to CHROMagar ECC agar. Multiple media were used for the isolation of E . coli in order to test the efficacy of each media.

https://doi.org/10.1371/journal.pone.0197005.t001

E . coli colony count results for each season is shown in Fig 2 in log 10 CFU/100 ml. Approximately 39% (177/458) of the total samples exceeded the United States Environmental Protection Agency (U.S. EPA) threshold for recreational activities, which is 235 CFU/100 ml based on a one-time measurement [ 1 ]. The average of the E . coli counts per season was above the threshold during six out of the eight sampling seasons, while the median E . coli counts exceeded the threshold only in Spring and Summer seasons of 2016. The E . coli counts were as low as undetectable (detection limit of 50 CFU/100 ml) and as high as 1.2×10 4 CFU/100 ml. The number of sampling sites that exceeded 235 CFU/100 ml for each sampling event is shown in Table 2 .

X-axis represents each sampling season with the numbers in parenthesis indicating the total number of water samples. Y-axis represents the E . coli counts in log 10 CFU/100 ml. The threshold represents the EPA threshold for water quality for recreational purposes.

https://doi.org/10.1371/journal.pone.0197005.g002

https://doi.org/10.1371/journal.pone.0197005.t002

Identification and characterization of E . coli

Phylo-groups and diarrheagenic pathotypes of E . coli isolates recovered from surface water are shown in Tables 3 and 4 . Using the quadruplex phylo-typing method, six phylo-groups (A, B1, B2, C, E, and F) were identified while three isolates could not be assigned a phylo-group (unknown; U). The most prevalent groups were B2 (31.7%; 157/496) and B1 (30.8%; 153/496). Fewer isolates were identified as groups E (23.2%; 115/496), A (6.7%; 33/496), F (4.6%; 23/496), and C (2.4%; 12/496).

https://doi.org/10.1371/journal.pone.0197005.t003

https://doi.org/10.1371/journal.pone.0197005.t004

Out of a total of 496 E . coli isolates, 19 EPEC and 1 STEC, positive for stx2 , were detected ( Table 4 ). The STEC isolate did not belong to serogroup O157 or O111. The majority of EPEC (15/19) isolates belonged to phylo-group B2 while the STEC belonged to phylo-group B1. No EAEC, EIEC, EHEC, or ETEC were detected. Locations where EPEC and the STEC were isolated are indicated on Fig 1 .

Most of the E . coli isolates were susceptible to the 14 drugs tested with only 6.9% (34/496) of the isolates exhibiting resistance to any of the drugs. These 34 AR E . coli were isolated from 24 sampling sites; eight of the sites had two AR E . coli isolated from them, and one site had three AR E . coli isolated from it. For this study, we considered resistance to two or more classes of antimicrobials as multidrug resistance (MDR). We chose this cut off to indicate resistance to multiple classes of antimicrobials rather than resistance to multiple antimicrobials, which if in the same class could be conferred by a single gene or genetic mutation. Therefore, using this definition helps to indicate that an isolate which is resistant to multiple classes of antimicrobials may have multiple mechanisms of AR. MDR was observed in 15 of the isolates. Eleven different MDR patterns were detected, including one isolate resistant to seven antimicrobials ( Table 4 ). Resistance to all of the 14 drugs tested was observed in the E . coli isolates from this study. Resistance to tetracycline was the most prevalent (76.5%; 26/34), followed by resistance to ampicillin (32.4%; 11/34), streptomycin (23.5%; 8/34), sulfisoxazole (23.5%; 8/34), and nalidixic acid (14.7%; 5/34). Locations from which AR E . coli were isolated are shown on Fig 1 . Interestingly, none of the EPEC or STEC isolates was resistant to any of the antimicrobials tested.

Prevalence of E . coli in the watershed

The results of this study indicated that E . coli was highly prevalent in the Upper Oconee Watershed as E . coli was isolated from almost every water site sampled each season. Due to the ubiquity of E . coli , no seasonal variations in presence was detectable; however, colony counts did vary. E . coli colony counts were determined using the 3M ™ Petrifilm ™ method, which is often used for volunteer-based water quality monitoring for its effectiveness, cost efficiency, and simplicity of use and storage [ 25 – 27 ]. Consistent with the previous reports on the water quality of the Upper Oconee Watershed [ 11 , 14 – 16 ], high E . coli counts were detected in the present study, which is evidence for widespread fecal contamination within the watershed. The E . coli counts of the water samples often exceeded the EPA threshold for recreational activities such as swimming and water skiing, which is 235 CFU/100 ml based on a one-time measurement [ 1 ]. The E . coli counts exceeded the threshold more often in the spring and summer seasons than in the fall and winter seasons, likely due to warmer water temperatures supporting growth of this enteric bacterium. In general, rural streams had acceptable E . coli counts while urban and suburban streams had higher levels of E . coli counts, which may be attributed to surface runoff from built infrastructure, leaking sewer lines, and failing septic systems. E . coli counts exceeding 10 3 CFU/100 ml were frequently observed which warrant special attention as it may indicate direct sewage contamination [ 25 ].

Pathogenic potential of E . coli in the watershed

Phylo-grouping PCR results showed that a third of all the E . coli isolates belonged to phylo-group B2, which is known to be associated with virulence and accounts for the majority of extra-intestinal infections [ 28 ]. The second most prevalent group was B1, to which commensal E . coli typically belong [ 28 ]. None of the isolates belonged to phylo-group D, which was contrary to previous reports that have shown a sizeable percentage of the group D isolates in environmental water samples [ 29 – 31 ]. The percentages of E . coli isolates that belonged to phylo-group D were 25.0% in the Mid-Atlantic region of the U.S. [ 29 ], 10.8% in the Yeongsan River basin of South Korea [ 30 ], and as high as 80.0% in the St. Clair River and Detroit River [ 31 ]. However, as opposed to the previous studies that used a triplex PCR developed by Clermont et al . in 2000 [ 32 ], the current study used a quadruplex PCR which was developed by Clermont et al . in 2012 as an improvement of the previous PCR method [ 18 ]. With the refined knowledge of E . coli phylogenetic group structure using multi-locus sequence type (MLST) data, new phylo-groups C, E, F, and Escherichia clade I were recognized and included in the phylo-typing PCR method, demonstrating the significant percentage of incorrect phylo-group assignment of E . coli strains using the previous triplex PCR [ 18 , 33 ]. Unfortunately, few studies in the literature have yet to use the quadruplex PCR method to characterize E . coli isolates from surface water. Therefore, it is unclear if it is the use of the different methods that has resulted in the difference in the percentages of the phylo-groups or if the phylo-groups follow a region- or site- specific pattern.

EPEC was rarely isolated in water samples in this study, with a total of 19 EPEC isolates detected. Humans are the main reservoir of EPEC, which causes watery diarrhea primarily in children under two years old [ 2 ]. Although this strain of E . coli persists in developing countries as a cause of diseases [ 2 ], EPEC is no longer a public threat in developed countries and only 30 cases of EPEC infections were confirmed in the U.S. from 2014 to 2016 [ 2 , 34 – 36 ]. A majority of EPEC belonged to phylo-group B2, which was consistent with previous studies that have reported that B2 strains tend to harbor more virulence determinants than the strains that belong to other phylo-groups [ 37 – 39 ]. Only one STEC was detected in any water sample during any season in the present study. Because this stx2 -positive isolate does not have any other virulence factor, such as eaeA , and cattle are known to be a vast reservoir of STEC [ 3 ], it is very probable that this STEC isolate originated from an animal source. E . coli O157, responsible for most human infections among the STECs in developed countries [ 3 , 21 ], has often been detected from surface water [ 40 , 41 ]. E . coli O157 outbreaks involving surface water contaminated with human and animal feces have been previously documented as well [ 5 , 7 , 9 ]. However, the present study did not detect any E . coli O157 isolates. While EAEC, EHEC, EIEC, and ETEC have been previously identified in surface water [ 42 ], none were detected in the Upper Oconee Watershed similar to findings for the St. Clair and Detroit rivers [ 31 ]. Locations from which the EPEC and STEC isolates were collected are indicated in Fig 1 . Overall, not many diarrheagenic strains of E . coli have been identified from the Upper Oconee Watershed; nevertheless, the isolation of EPEC and STEC from the surface water does suggest potential exposure of environmental water to fecal contamination of human and/or animal origin.

Antimicrobial resistant E . coli in the watershed

Only a small portion of water samples harbored E . coli resistant to any of the 14 drugs tested. However, it is important to note that our isolation method did not use antimicrobials to select for resistant strains; therefore, the level of 6.9% resistant E . coli likely represented a true level of resistant E . coli in the watershed, which is not trivial considering the high level of some of the sample colony counts. Resistance was observed most often to tetracycline, followed by ampicillin, streptomycin, sulfisoxazole, and nalidixic acid. The high resistance rate to tetracycline has been previously reported in other studies [ 43 – 45 ], indicating that the resistance to tetracycline is prevalent in environmental water. This observation was expected as tetracycline is one of the most widely used antimicrobials for treatment of human and animal infections as well as the historic use for agricultural purposes as growth promoters [ 46 , 47 ]. The frequency of resistance to the antimicrobials listed above corresponded with findings from other regions, while the prevalence of AR in E . coli from the Upper Oconee Watershed was less than expected based on levels seen in other environmental water sources [ 38 , 43 – 45 ]. High levels of AR to a variety of antimicrobials have been reported for E . coli isolates from aquatic environment, as high as 82% in other regions of the country [ 38 , 43 – 45 ], and as high as 100% in other parts of the world [ 48 – 50 ]. The difference in the level of AR with the E . coli isolates from this study may be partially due to the antimicrobial drugs chosen for testing. Variances in the therapeutic drugs used and the levels of fecal contamination may also have contributed to the difference.

AR E . coli were mostly recovered from residential areas. The exact locations of the sites with the GPS coordinates are in S1 Table and locations where AR positive samples were collected are indicated in Fig 1 . The city of Athens, which encompasses Clarke County and some parts of Oconee and Jackson Counties, is served by a sewer system with surprisingly high cases of sewage problems [ 51 ]. These include the case of an unknown amount of improperly treated wastewater being discharged into a creek over an unknown period of time [ 52 ], increasing the likelihood of isolating AR E . coli of human source within the residential land areas. A few other sites from where AR E . coli were recovered were located near agricultural operations i.e. MIDO 103, MIDO 305, MIDO 505, and MIDO 507. There were cattle pastures, a poultry farm, and a small horse farm near the sampling sites which could have been potential sources of AR E . coli isolated.

McNutt Creek flows through suburban residential and commercial areas of Athens. Several sampling sites were located along the creek and its tributaries, and the quality of water has been shown to be a concern due to high E . coli counts and the presence of AR and pathogenic E . coli . Four out of 19 EPEC isolates and six out of 34 AR E . coli isolates were collected from McNutt Creek alone. McNutt Creek is on the EPA Total Maximum Daily Load (TMDL) 303(d) list of Impaired Waters in terms of fecal coliform due to nonpoint sources and urban runoff [ 53 ]. Continuous monitoring of the creek to track the sources of contamination is required to gather data for the improvement of the creek’s water quality.

Although there are several papers on E . coli from surface waters of different locations around the world, relatively little has been studied about E . coli prevalence, pathotypes, and antimicrobial susceptibility in surface waters of a mixed-use watershed in the U.S. One of the most similar studies was reported by Ibekwe et al . who collected samples from 20 sites quarterly over a twelve-month period from the middle Santa Anna River in Southern California, USA [ 45 ]. However, the watershed drained by that river is dominated by a large area of cattle farms and discharges from three wastewater treatment plants. In addition, that study also sampled sediments thus representing the environment in and around the river, whereas our study focused on bacteria within the moving water column indicative of what residents would be exposed to by recreational, agricultural, and municipal use of surface water. Our study area was diverse and represented land use from the undeveloped forest, agricultural and rural residential lands (about 62% of the land in the watershed) to densely developed industrial and suburban- residential lands (about 38%). Water samples were collected quarterly for two years from 100 different sampling sites that encompassed the entire watershed, incorporating not only relatively pristine streams but also streams with a history of human impacts, such as runoff from agricultural activities as well as contaminated effluents from wastewater treatment plants, discharges from failing septic systems, and sewer line leaks. As approximately half of the U.S. population lives in suburban areas [ 54 ], this mixed-use watershed may be a good representation of the conditions many U.S. residents are exposed to by surface water used for recreational, agricultural, and municipal purposes.

This study has shown the seasonal and spatial prevalence and characteristics of E . coli in surface water of the Upper Oconee Watershed, Athens, GA, including the presence of pathogenic and AR E . coli . E . coli resistant to therapeutic drugs were not highly prevalent in the environment and these commensal bacteria may not appear to be a risk to public health. However, E . coli is known to harbor AR genes on plasmids, transposons, and integrons, and these mobile genetic elements can be transferred between organisms of the same species or different genera through horizontal gene transfer [ 55 , 56 ]. Therefore, further studies are required to assess risks associated with E . coli harboring AR genes and the potential of transferring these genes to other bacteria, including commensal E . coli and other bacterial pathogens which could have a detrimental impact on public health.

Supporting information

S1 table. master file of the e . coli isolated from the upper oconee watershed..

a amoxicillin/clavulanic acid (Amo), ampicillin (Amp), azithromycin (Azi), cefoxitin (Fox), ceftiofur (Tio), ceftriaxone (Axo), chloramphenicol (Chl), ciprofloxacin (Cip), gentamicin (Gen), nalidixic acid (Nal), streptomycin (Str), sulfisoxazole (Sul), tetracycline (Tet), trimethoprim/sulfamethoxazole (Tri). b EPEC: enteropathogenic E . coli and STEC: Shiga toxin-producing E . coli .

https://doi.org/10.1371/journal.pone.0197005.s001

Acknowledgments

The authors gratefully acknowledge Elizabeth A. Ottesen and Erin K. Lipp for helpful discussions. We thank Jacob M. Mcdonald for the map of the UOWN sampling sites. We also thank the Ottesen laboratory personnel, Bruno Giri, Phillip Bumpers, the UOWN science and monitoring committee, and the UOWN volunteers for their assistance in collecting the water samples. The mention of trade names or commercial products in this manuscript is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

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The diversity of escherichia coli pathotypes and vaccination strategies against this versatile bacterial pathogen.

1. Introduction

1.1. escherichia coli, 1.2. phenomenon of antibiotic resistance, 2. diarrheagenic e. coli pathotypes, 2.1.1. molecular pathogenesis, 2.1.2. vaccine strategies against epec, 2.2.1. molecular pathogenesis, 2.2.2. vaccine strategies against ehec.

2.3.1. Molecular Pathogenesis

2.3.2. vaccine strategies against etec, 2.4.1. molecular pathogenesis, 2.4.2. relationship with shigella, 2.4.3. vaccine strategies against eiec/shigella, 2.5. eaec and daec, 2.5.1. molecular pathogenesis, 2.5.2. vaccine strategies against eaec and daec, 2.6.1. molecular pathogenesis, 2.6.2. relation of aiec and crohn’s disease, 2.6.3. insights for vaccine development, 3. extraintestinal pathogenic e. coli, 3.1. uropathogenic e. coli (upec), 3.1.1. urinary tract infection (uti), 3.1.2. molecular pathogenesis of upec, 3.1.3. vaccine strategies against upec, 3.2. mnec, sepec, vaccine strategies against mnec, 4. pathogenic e. coli of importance to animal health, 4.1. avian pathogenic escherichia coli (apec), 4.1.1. apec pathogenesis, 4.1.2. vaccines and vaccination strategies against apec, 4.2. porcine colibacillosis, 4.2.1. resistance to multiple antibiotics in e. coli causing diarrhea in swine, 4.2.2. need of vaccines and vaccine strategies against porcine pathogenic e. coli, 4.3. bovine colibacillosis, 4.4. mastitis in cattle and swine, 5. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, conflicts of interest.

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Pokharel, P.; Dhakal, S.; Dozois, C.M. The Diversity of Escherichia coli Pathotypes and Vaccination Strategies against This Versatile Bacterial Pathogen. Microorganisms 2023 , 11 , 344. https://doi.org/10.3390/microorganisms11020344

Pokharel P, Dhakal S, Dozois CM. The Diversity of Escherichia coli Pathotypes and Vaccination Strategies against This Versatile Bacterial Pathogen. Microorganisms . 2023; 11(2):344. https://doi.org/10.3390/microorganisms11020344

Pokharel, Pravil, Sabin Dhakal, and Charles M. Dozois. 2023. "The Diversity of Escherichia coli Pathotypes and Vaccination Strategies against This Versatile Bacterial Pathogen" Microorganisms 11, no. 2: 344. https://doi.org/10.3390/microorganisms11020344

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• 1 University of California San Diego
• 2 UC San Diego
• PMID: 33231968
• Bookshelf ID: NBK564298

Escherichia coli ( E. coli ) is a gram-negative bacillus known to be a part of normal intestinal flora but can also be the cause of intestinal and extraintestinal illness in humans. There are hundreds of identified E. coli strains, resulting in a spectrum of disease from mild, self-limited gastroenteritis to renal failure and septic shock. Its virulence lends to E. coli’s ability to evade host defenses and develop resistance to common antibiotics. This review will divide E. coli infections into those causing intestinal illness and those causing extraintestinal illness.

Intestinal illnesses will be described by the causative E. coli subtypes, including enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), which is also known as Shiga toxin-producing Escherichia coli (STEC) and will be referred to as EHEC/STEC, enteroinvasive Escherichia coli (EIEC), enteropathogenic Escherichia coli (EPEC), and enteroaggregative Escherichia coli (EAEC). Extraintestinal illnesses will be described based on clinical disease.

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Conflict of interest statement

Disclosure: Matthew Mueller declares no relevant financial relationships with ineligible companies.

Disclosure: Christopher Tainter declares no relevant financial relationships with ineligible companies.

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• DOI: 10.5772/67390
• Corpus ID: 90047418

Isolation and Characterization of Escherichia coli from Animals, Humans, and Environment

• A. M. Lupindu
• Published 12 July 2017
• Environmental Science, Biology, Medicine

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Study on bacteria isolates and antimicrobial resistance in wildlife in sicily, southern italy, genomic characterization of fecal escherichia coli isolates with reduced susceptibility to beta-lactam antimicrobials from wild hogs and coyotes, characterization of foodborne pathogens and enterotoxigenic staphylococcus aureus isolates with detection of antibiotic resistance from beef meat, assessing the effect of oxytetracycline on the selection of resistant escherichia coli in treated and untreated broiler chickens, molecular epidemiology of mcr-1, blakpc-2, and blandm-1 harboring clinically isolated escherichia coli from pakistan, antimicrobial sensitivity of shiga toxin-producing escherichia coli (stec) and virulence genes of representative isolates in port harcourt, nigeria, isolation and antimicrobial susceptibility pattern of escherichia coli from laying chicken and egg in jimma town, ethiopia, occurrence and antimicrobial susceptibility patterns of escherichia coli and escherichia coli o157 isolated from cow milk and milk products, ethiopia, processed ready-to-eat (rte) foods sold in yenagoa nigeria were colonized by diarrheagenic escherichia coli which constitute a probable hazard to human health, prevalence, antimicrobial resistance, and pathogenic potential of enterotoxigenic and enteropathogenic escherichia coli associated with acute diarrheal patients in tangail, bangladesh., 51 references, 2 serotyping of escherichia coli, isolation and characterization of verocytotoxin-producing escherichia coli o157 strains from dutch cattle and sheep, characterization of enteroaggregative escherichia coli (eaec) clinical isolates and their antibiotic resistance pattern., comparative genomic indexing reveals the phylogenomics of escherichia coli pathogens, comparison of dna hybridization and pcr assays for detection of putative pathogenic enteroadherent escherichia coli, diffusely adherent escherichia coli strains isolated from children and adults constitute two different populations, what defines extraintestinal pathogenic escherichia coli, overview of molecular typing methods for outbreak detection and epidemiological surveillance., differentiation between shigella, enteroinvasive escherichia coli (eiec) and noninvasive escherichia coli, extraintestinal pathogenic and antimicrobial-resistant escherichia coli contamination of 56 public restrooms in the greater minneapolis-st. paul metropolitan area, related papers.

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Escherichia Coli: What Is and Which Are ?

Marta cobo-simón.

Department of Molecular Biosciences, University of Texas at Austin, Austin, TX

Howard Ochman

Associated data.

All complete genomes used in this analysis are available from the NCBI database ( https://www.ncbi.nlm.nih.gov/ ) using accession numbers listed in supplementary table S1, Supplementary Material online.

Escherichia coli have served as important model organisms for over a century—used to elucidate key aspects of genetics, evolution, molecular biology, and pathogenesis. However, defining which strains actually belong to this species is erratic and unstable due to shifts in the characters and criteria used to distinguish bacterial species. Additionally, many isolates designated as E. coli are genetically more closely related to strains of Shigella than to other E . coli , creating a situation in which the entire genus of Shigella and its four species are encompassed within the single species E. coli . We evaluated all complete genomes assigned to E. coli and its closest relatives according to the biological species concept (BSC), using evidence of reproductive isolation and gene flow (i.e., homologous recombination in the case of asexual bacteria) to ascertain species boundaries. The BSC establishes a uniform, consistent, and objective principle that allows species-level classification across all domains of life and does not rely on either phenotypic or genotypic similarity to a defined type-specimen for species membership. Analyzing a total of 1,887 sequenced genomes and comparing our results to other genome-based classification methods, we found few barriers to gene flow among the strains, clades, phylogroups, or species within E. coli and Shigella . Due to the utility in recognizing which strains constitute a true biological species, we designate genomes that form a genetic cohesive group as members of E. coli BIO .

Introduction

When initially isolated, Escherichia coli was designated Bacillus coli communis , a latinization describing its prominent characteristic as a “common colon bacterium” that could be readily cultured in a variety of substrates. The original specimen, as first described in 1885, was distinguished by its colony and cellular morphology, and its ability to ferment glucose, produce acid, and sour milk ( Escherich 1885 ). Upon its rechristening in 1,919 to acknowledge its discoverer, and in the decades that ensued, features used for assignment to this species were expanded to include a suite of characters that distinguish E. coli from other enteric species ( Koser 1923 ; Kauffmann 1944 ). Most notably, E. coli are lactose, catalase, and indole positive, and oxidase, urease, and citrate negative, although there is a low level of polymorphism for many of these properties.

Genetic and genomic features entered into the classification of E. coli in the 1960s with the application of DNA–DNA hybridization (DDH) procedures ( Marmur et al. 1963 ). By this method, strains were considered as members of E. coli if they displayed ≥70% DNA similarity to the reference strains ( Brenner et al. 1972 )—noting that although DDH percentages do not match the actual amount of DNA identity between strains ( Rosselló-Mora 2006 ), this method pioneered a threshold-based approach for defining bacterial species. Subsequently, other nucleic-acid-based cutoffs were applied to the delineation of bacterial species, such as ≥97% ( Tindall et al. 2010 ; Yarza et al. 2014 ) and more recently ≥99% ( Edgar 2018 ) 16S RNA sequence identity, or ≥95% average nucleotide identity (ANI) ( Konstantinidis and Tiedje 2005 ) for the core set of genes shared among strains ( Jain et al. 2018 ). Naturally, there is a certain circularity to this approach since sequence-identity thresholds were ascertained from strains that were already assigned to E. coli based on metabolic, morphological, or biochemical features, thereby constraining the genetic cutoffs to species boundaries that were already established. And unfortunately, hybridization and sequence-identity thresholds are convenient rather than universal, their biological basis remains unclear.

Phylogenetic analysis of E. coli strains that were considered to span the diversity in the species at large defined six main clades (A, B1, B2, D, E, and F) and several rarer clades ( Herzer et al. 1990 ; Chaudhuri and Henderson 2012 ). However, expanding the set to include strains from additional animal and environmental sources yielded five “cryptic” clades (termed CI to CV) that were all more closely related to E. coli than to its sister species Escherichia fergusonii ( Walk et al. 2009 ; Luo et al. 2011 ). The taxonomic status of these five unclassified clades remains uncertain: they cannot be differentiated from E. coli based on phenotypic characters, but they are genetically divergent, which led to a proposal that a least some of these clades (e.g., Clades III + IV and Clade V) might represent distinct species ( Walk 2015 ).

As additional full genomes were integrated into the analyses, the phylogenetic structure and evolutionary relationships of E. coli became more refined, with recognition of increased numbers of subspecific groups ( Lu et al. 2016 ; Abram et al. 2021 ) and suggestions that some might represent actual or incipient species ( Didelot et al. 2012 ; Kang et al. 2021 ). To accommodate the burgeoning numbers of sequenced strains in all taxa, the Genome Taxonomy Database (GTDB; gtdb.ecogenomic.org/) recommended the application of a genome-wide identity threshold (analogous to ANI) to define bacterial species ( Parks et al. 2018 ). Imposing their metrics, strains currently classified as E. coli would be split into six species— E. coli , E. coli _E, Escherichia ruysiae , Escherichia marmotae, Escherichia sp001660175 , and Escherichia sp005843885 —with the majority consigned to E. coli ( Parks et al. 2021 ).

Classification of E. coli has also been confounded by the intransigence of Shigella as a separate genus. Every strain assigned to Shigella appears to fall within the variation spanned by E. coli ( Brenner et al. 1973 ; Ochman et al. 1983 ), and the four Shigella species originated independently, and multiple times, from within E. coli ( Rolland et al. 1998 ; Pupo et al. 2000 ; Lan and Reeves 2002 ). Clearly, the taxonomy of E. coli is idiosyncratic and often supports conflicting results. To resolve these incongruencies, and to apply consistent and objective criteria to identifying species boundaries, we analyze and classify a comprehensive set of Escherichia and Shigella genomes according to the biological species concept (BSC) ( Mayr 1942 ), a universally accepted procedure that circumscribes species based on homologous gene exchange. Although asexuality is often assumed to render bacteria immune to classification by the BSC ( Donoghue 1985 ; Rosselló-Mora and Amann 2001 ; Costechareyre et al. 2009 ), the patterns of recombination in E. coli and related enteric bacteria provide a consistent and robust signal for species assignment ( Brenner and Falkow 1971 ; Shen and Huang 1986 ; Dykhuizen and Green 1991 ; Lawrence and Retchless 2009 ; Didelot et al. 2012 ) and allow the application of a single biological feature to define species across all branches of the Tree of Life.

To establish the species boundaries of E. coli and determine which sequenced genomes should be assigned to this species, we implemented three genome-based methods, including two ( ConSpeciFix and PopCOGenT ) that adhere to the precepts of the BSC. We considered 1,635 complete genomes designated as E. coli in the National Center for Biotechnology Information (NCBI) database ( www.ncbi.nlm.nih.gov/ ) as of August 2020. To ensure that the breadth of variation in the species at large is represented, we included the genomes of strains classified to the five Escherichia phylogroups (Clades I–V) described by Walk et al. (2009) , to the E. coli phylogroups resolved by Abram et al. (2021) , and to the newly designated Escherichia species proposed by the GTDB ( E. albertii, E. coli , E. coli_ E, E. fergusonii, E. marmotae, E. ruysiae, Escherichia sp001660175, Escherichia sp002965065, Escherichia sp004211955, and Escherichia sp005843885) (gtdb.ecogenomic.org). Additionally, we analyzed all other fully sequenced genomes assigned to the genus Escherichia as well as representatives of the four designated species of Shigella , which are known to have originated from within E. coli but have mostly maintained their status as a separate genus for historical reasons.

ConSpeciFix

Using gene flow as a condition for species membership, this method calculates recombination based on homoplasies in genes common to the strains under consideration ( Bobay et al. 2018 ).

Assignment of sequenced genomes to the biological species E. coli BIO . Wedges are labeled according to their taxon designation in the NCBI database or their assignment to an Escherichia phylogroup by Walk et al., with the number of genomes in each taxon indicated. Note that genomes that are both assigned to an Escherichia phylogroup (CI–CV) and taxonomically defined in the NCBI are excluded from counts of NCBI genomes. For example, one of the genomes assigned to E. coli by NCBI but excluded from E. coli BIO belongs to Walk Clade IV and was therefore excluded from the count of E. coli .

Maximum-likelihood phylogenetic tree of selected Escherichia genomes. Genomes were selected to represent the extent of diversity present in the genus and have <99.8% ANI to their nearest relative. For each genome, strain accession number and designation in the NCBI database is followed, from left to right, by PopCOGenT species-group, GTDB v207 classification, Walk et al. phylogroup (Clades I–V) and Abram et al. phylogroups wherever possible, ANI to E. coli ATCC 11775, and membership status in E. coli BIO . (Note that GCA_002109985_1, E. marmotae E1118, is labeled as E. coli in the PATRIC database). PopCOGenT species-groups are distinguished by number, and ANI % denotes extent of sequence identity to the reference genome (marked with asterisk): the first 24 genomes in the tree present an ANI > 97%; the following 30 an ANI between 95 and 97%, and the last 16 an ANI <95%. All branches have bootstrap support values >90% except for the strains GCA_000007445.1, GCA_000488315.1, GCA_000459455.1 and GCA_002911335.1, which are <60%. Seven of the 12 NCBI-classified E. coli strains that were excluded from E. coli BIO are NCBI pathogen detection assemblies (i.e., surveillance genomes) and were not classified by the GTDB classification: those genomes lacking taxonomic assignation in GTDB were classified to the same species as their closest relative having an ANI >95%.

• (ii) Applying ConSpeciFix to assess species status of phylogroups defined by Walk et al. (2009) and Abram et al. (2021) . All the studied strains classified to the E. coli phylogroups of Abram et al., and to the E. coli taxonomic group and Clade I specified by Walk et al., which together include strains classified as E. coli and Escherichia sp. in NCBI, are members of E. coli BIO based on ConSpeciFix . Genomes in their remaining clades (Clades II–V) constitute different species by ConSpeciFix , with the exception of one genome in Clade IV ( E. coli B49-2 serovar O157:H7) and one in Clade V ( E. coli strain E620 serovar ON5), both of which are members of E. coli BIO ( supplementary table S1, Supplementary Material online).
• (iii) Applying ConSpecFix to assess the status of E. coli species defined by GTDB . The GTDB recognizes E. coli and nine additional species ( E. ruysiae , E. marmotae, E. coli_ E . E. fergusonii, Escherichia albertii, Escherichia sp001660175 , Escherichia sp002965065 , Escherichia sp004211955, and Escherichia sp005843885). Of these nine additional species, only Escherichia coli _E, one strain of Escherichia sp001660175 (based on ANI), three strains of Escherichia sp005843885 (one of them based on ANI), and three strains of E. ruysiae (two of them based on ANI) were classified as E. coli in the NCBI database ( supplementary table S1, Supplementary Material online). Although listed as differently by the GTDB, one strain of E. albertii and one strain of E. marmotae are classified as E. coli in the NCBI. Of the 12 genomes assigned to E. coli in the NCBI but excluded from E. coli BIO , nine were also excluded from E. coli by the GTDB ( supplementary table S1, Supplementary Material online). ConSpeciFix assigned the GTDB species Escherichia sp001660175 ( n = 1), sp004211955 ( n = 2), and sp005843885 ( n = 38) to a separate biological species. No members of these three GTDB species belong to E. coli BIO when used as test lineages, but they form a biological species distinct from E. coli BIO when Escherichia sp005843885 is used as a reference lineage. None of the other GTDB species is a member of either E. coli BIO or this new species.
• (iv) Other enteric species . All tested genomes of the four Shigella species are members of E. coli BIO . In contrast, none of the genomes currently classified to any of the other Escherichia species [ E. albertii ( n = 1) , E. fergusonii ( n = 2), E. marmotae ( n = 1)] or to any of the other enteric genera considered [ Proteus ( n = 2) , Citrobacter ( n = 2) , Cronobacter ( n = 2) , Salmonella ( n = 111) , Enterobacter ( n = 6), and Klebsiella ( n = 4)] is a member of E. coli BIO . Genomes from genera other than Escherichia were included as controls.

PopCOGenT is an alternate method for grouping genomes based on gene flow ( Arevalo et al. 2019 ). For the representative set of genomes evaluated by this method ( n = 128), there were a total of 21 species-groups, of which 10 contained strains designated as E. coli in the NCBI database ( supplementary table S1, Supplementary Material online). The phylogenetic relationships of a dereplicated subset of these genomes, along with their nomenclature, strain and species designations in different databases, and species-groupings based on several metrics, are presented in figure 2 .

• (i) Applying PopCOGenT to assess species status of clades defined by Walk et al. (2009) and Abram et al. (2021) . Genomes from the E. coli taxonomic group specified by Walk are assigned to PopCOGenT species-groups 0 and 1 ( fig. 2 ; supplementary table S1, Supplementary Material online), and Clades I, II, and III of Walk are each classified as different PopCOGenT species-groups (4, 5, and 6, respectively). Genomes from Walk Clade IV assort into two species-groups: one of which contains only Clade IV genomes, and another that contains genomes from both Clade V and the canonical E. coli and Shigella flexneri taxonomic groups specified by Walk. Similarly, genomes from Clade V of Walk segregate into two species-groups—the aforementioned one that contains genomes from Clade IV and the canonical E. coli and S. flexneri taxonomic groups, and a unique species-group (19) that contains only Clade V genomes. Several of the E. coli phylogroups defined by Abram et al. were distinguished as different species-groups by PopCOGenT .
• (ii) Applying PopCOGenT to assess the status of E. coli species defined by GTDB . The five GTDB-recognized species within E. coli ( E. coli, E. coli_ E , E. ruysiae, Escherichia sp001660175, and Escherichia sp005843885) and the five other Escherichia species ( E. albertii , E. fergusonii, E. marmotae, E. sp002965065, and E. sp004211955) were classified to multiple species-groups by PopCOGenT ( supplementary table S1, Supplementary Material online). Each of the Escherichia species recognized by the GTDB forms a unique PopCOGent species-group, except 1) E. ruysiae, whose members were distributed into two PopCOGent species-groups (6, 15), 2) E. coli , whose members were distributed into four PopCOGent species-groups (0, 1, 2, 4), and 3) E. coli _E, whose members were distributed into two PopCOGent species-groups (8, 17) ( fig. 2 ; supplementary table S1, Supplementary Material online).
• (iii) Other enteric species . Whereas PopCOGenT separated the NCBI-designated strains of E. coli into 10 species-groups, all Shigella genomes considered by PopCOGenT , except Sh. dysenteriae (accession GCA_002950055.1), were classified as members of species-group 0. Most strains that were assigned to Escherichia species other than E. coli (or whose species status went unassigned) were deemed separate species by PopCOGenT , although many partitioned in species-groups that also contained members of E. coli . Though not included in figure 2 , PopCOGenT distinguished Salmonella enterica , Salmonella bongori , Enterobacter cloacae, Enterobacter carcerogenus , and Proteus mirabilis as distinct species.

This metric is based on sequence-identity thresholds (typically 95%) to delineate strains that constitute a species ( Jain et al. 2018 ).

• (i) Applying ANI to assess species status of clades defined by Walk et al. (2009) and Abram et al. (2021) . Genomes from S. flexneri and the E. coli taxonomic group specified as Clade I by Walk, and one genome each from Clades IV and V, are all classified as members of the same species based on 95% ANI to the reference genome, E. coli ATCC 11775. Applying this 95% ANI threshold, the studied phylogroups distinguished by Abram et al. are also included in this species ( supplementary table S1, Supplementary Material online). All genomes in this ANI species were originally designated as E. coli or S. flexneri in NCBI, except in the case of one genome classified as Escherichia sp. All remaining members of Clades IV and V, and all other members of the other clades defined by Walk et al., are sufficiently distant from E. coli ATCC 11775 and are not considered members of the species at this ANI threshold.
• (ii) Applying ANI to assess the status of E. coli species defined by GTDB . Because the GTDB circumscribes species based on sequence-identity thresholds, the majority of the genomes assigned to E. coli have an ANI > 95% to the E. coli ATCC 11775 reference genome; however, there are a few exceptions due to the normalization applied by this database ( supplementary table S1, Supplementary Material online).
• (iii) Other enteric species . applying a sequence-identity threshold of 95% to E. coli ATCC 11775, all tested genomes of the four Shigella spp. are members of E. coli. None of the genomes classified to other Escherichia species ( E. albertii and E. fergusonii ) or to any of the other enteric genera ( Proteus, Citrobacter, Cronobacter, Salmonella, Enterobacter, and Klebsiella ) is a member of E. coli at this sequence-identity threshold.

Applying the many-to-many option in ANI returned results that were virtually identical to those recovered with the one-to-many comparisons to the single reference genome. For example, all other enteric species yielded ANI values <95% to members of both E. coli and Shigella . With regard to the biological species ( E. coli BIO ) defined ConSpeciFix , most genomes displayed ANI values <95% using the many-to-many option; however, the minimum ANI of 93.90% occurred between two strains having 97% and 98% ANI with the reference genome.

Maximum-Likelihood (ML) Phylogeny

To examine the evolutionary relationships among strains, we constructed a phylogeny on the dereplicated set of 70 genomes having <99.8% ANI to one another.

• (i) Applying an ML phylogeny to assess species status of clades defined by Walk et al. (2009) and Abram et al. (2021) . Our results broadly confirm the phylogroups distinguished by Walk et al. (2009) , which is not surprising given that their phylogroups represent phylogenetically resolved clades. All E. coli and Shigella genomes that they defined were monophyletic, and Clades I, II, III, IV, and V each formed monophyletic groups, with the exception of one strain from each of Clade IV and Clade V, which grouped with E. coli and Shigella . In our tree, the clade containing E. coli and Shigella is most closely related to Walk Clade I, which is a sister group to E. fergusonii , and Walk Clades III, IV, and V together form a separate clade. In addition, each of the phylogroups resolved Abram et al. (2021) is monophyletic ( fig. 2 ).
• (ii) Applying an ML phylogeny to assess the status of E. coli species defined by GTDB . The clades defined in the ML phylogeny are consistent with the species distinguished by GTDB, and each is monophyletic. The only exceptions are the two strains classified as E. fergusonii , which reside on a very long branch, have low ANI (<95%) to the E. coli reference strain, and are not members of E. coli BIO based on ConSpeciFix ( fig. 2 ; supplementary table S1, Supplementary Material online). The high bootstrap support of this branch suggests an ancient separation followed by limited recombination with divergent members of E. coli , as exemplified by the inclusion of E. fergusonii genomes in PopCoGenet species-group 4.
• (iii) Other enteric species. Based on the ML phylogeny, the only members of other Escherichia species that occur in the monophyletic group that contains E. coli and Shigella are the two aforementioned strains of E. fergusonii .

Bacterial strains were originally typed as E. coli based on their growth characteristics and possession of specific metabolic properties, and, more recently, based on their sequence similarity to one another or to a canonical strain. In addition, there are sufficiently high levels of recombination among strains, despite their asexual mode of reproduction, to warrant the classification of strains to this species based on the BSC. Using homologous exchange as the sole criterion for species assignment, we found that the vast majority of strains currently designated as E. coli, or as any of the species of Shigella, are all members of a single biological species, which we term E. coli BIO . Species-level definitions for the genus Escherichia have already been described by Walk ( 2015 ) and by Denamur et al. ( 2021 ), who have recently proposed a dichotomy between E. coli sensu stricto and E. coli sensu lato . However, such a classification scheme is inadequate because it does not have a biological basis and it can be universally applied (and, moreover, the new species, E. coli sensu lato , does not include Shigella , which belongs to the same species based on all genetic-based methods).

The species boundaries of E. coli BIO , which are based solely on homologous recombination within the set of core genes shared by all strains, largely agree with the classifications proposed by other schemes. For example, all methodologies, except PopCOGenT , consider the E. coli phylogroups of Abram et al. (2021) as comprising a single species, whereas PopCOGenT separates them into multiple species. That PopCOGenT , which also uses gene flow to delineate species, distinguishes more species than ConSpeciFix is due to the fact that PopCOGenT considers entire genomes when assigning species membership and can include horizontally transferred regions that are confined to subsets (or even pairs) of strains. Given that events of horizontal gene transfer occur over broad phylogenetic distances (and even between organisms classified to different domains or kingdoms), we chose exclude regions that are sporadically distributed among genomes and to confine analyses to core genes present in all genomes considered.

Strains typed to Shigella have been viewed as distinct from E. coli because they exhibited certain defining characteristics, including the absence of motility (due to a deletion in the fliF operon or insertion in the flhD operon) ( Al Mamun et al. 1997 ) and an inability to ferment lactose (due to the lack of one or more lac fermentation or permease genes) ( Luria and Burrous 1957 ; Khot and Fisher 2013 ). Moreover, the four species of Shigella are conventionally distinguished from one another by their O serotypes ( Wheeler and Stuart 1946 ; Lan and Reeves 2002 ) because many of the other diagnostic properties, such as the utilization of mannitol and decarboxylation of ornithine, can be shared among species. However, the traits used to discriminate species of Shigella , and Shigella from E. coli , are often observed in enteroinvasive E. coli , which blurs the distinction between these species and genera.

In actuality, E. coli and Shigella were initially assigned to the same genus due to their similarities but to different species to distinguish pathogenic and nonpathogenic forms ( Bacillus dysenteriae and B. coli , respectively) ( Shiga 1898 ). But as chronicled in figure 3 , due to their medical significance, pathogenic strains were elevated to a separate genus in the following decades despite their resemblance to enteroinvasive E. coli ( Ewing et al. 1952 ). The close genetic relationship between E. coli and Shigella was initially recognized in the 1950s based on their ability to reciprocally recombine ( Luria and Burrous 1957 ), but because Shigella recombined with E. coli at lower frequencies than observed among strains of E. coli , each taxon maintained its status as a separate genus. However, subsequent analyses of genetic and genomic characters by DNA hybridization ( Brenner et al. 1969 , 1972 ), multilocus enzyme electrophoresis ( Ochman et al. 1983 ), and chromosomal and plasmid gene phylogenies ( Pupo et al. 2000 ; Lan and Reeves 2002 ) all indicated that strains typed as Shigella fall within the variation observed in E. coli .

Chronological changes in the S. dysenteriae and E. coli nomenclature. References used to produce this figure are listed in Supplementary material online.

The fact that Shigella remains classified as a distinct genus, despite its genetic and phenotypic overlap with E. coli, is further complicated by the fact that other named species within the genus Escherichia (e.g., E. albertii or E. fergusonii ) do not recombine with E. coli, and can be differentiated based on such metabolic characters as 1) the lack of acid production from D -xylose, melibiose, L -rhamnose, and dulcitol for E. albertii ( Hinenoya et al. 2019 ) and (2) an incapacity to ferment sorbitol and lactose, coupled with the ability to ferment adonitol, amygdalin, and cellobiose for E. fergusonii ( Farmer et al. 1985 ). Taken together, this creates a situation in which the genus Escherichia contains multiple distinguishable species, whereas the four named species of Shigella should be subsumed within E. coli .

To mitigate confusion that might stem from abolishing the genus Shigella , Brenner et al . ( 1973 ) proposed the use of two separate nomenclatures—one for diagnostic purposes and one for genetic purposes—though it is difficult to see how this serves as an improvement. Lan and Reeves ( 2002 ) regarded the species of Shigella as serotypes within E. coli and removed the generic name, referring to them simply as Boydii, Sonnei, Flexneri, and Dystenteriae. Meier-Kolthoff et al . ( 2014 ) proposed including the four Shigella species as subspecies of E. coli, with nomenclature following guidelines of the Bacteriological Code ( Lapage et al. 1992 ): In this system, for example, Shigella dysenteriae would be renamed as E. coli subsp. dysenteriae, and current members of E. coli as E. coli subsp. coli. Along similar lines, Parks et al . ( 2020 ) suggested including the four species of Shigella within the genus Escherichia , creating E. sonnei, E. boydii, E flexneri, and E. dysenteriae. However, based on DNA similarity threshold that they routinely use to define species, these newly named Escherichia species should remain within E. coli ( Parks et al. 2021 ).

To circumvent issues surrounding the elimination or amendment of species names, we propose that conspecifics defined by the BSC be classified under the heading of a single biological species, as denoted by a subscripted suffix “BIO” adjoined to the latin biome. This procedure would place strains of E. coli and Shigella under the umbrella of a single biological species, in this case, E. coli BIO , but would retain their full names to maintain clinically and historically relevant information. As such, S. dysenteriae would be labeled Ecoli BIO S. dysenteriae , and current members of E. coli as E. coli BIO followed by their strain designation. This resolution mimics the nomenclature developed for serovars of S. enterica and does not impose a taxonomic revision but is nevertheless useful in indicating which strains are members of the same biological species. The retention of strain appellations in the proposed scheme maintains consistency with the traditional nomenclature and avoids conflict with clinical identification and applications.

Despite the ability of E. coli and other bacteria to acquire genes from distant sources, recombination between shared homologs occurs primarily among sequences with high levels of similarity ( Shen and Huang 1986 ; Rayssiguier et al. 1989 ; Roberts and Cohan 1993 ; Matic et al. 1995 ; Zawadzki et al. 1995 ; Majewski and Cohan 1999 ). This feature enables a natural classification of bacteria into species based on their propensity for homologous exchange, a biological criterion that can be applied to all lifeforms. To assure the universality of species definition, it is, therefore, necessary to confine analyses of recombination to the core set of genes shared among genomes. Those sequences with rare or sporadic distributions, as might originate from infrequent or independent events of horizontal gene transfer between taxa, occur in eukaryotes as well as bacteria ( Akanni et al. 2015 ; Husnik and McCutcheon 2018 ; Wu et al. 2022 ), and can involve very distant taxa. Thus, such genes are best excluded from consideration when delineating species boundaries

Species, when defined by their capacity for gene flow, constitutes the only taxonomic rank based on a biological process rather than an arbitrary or subjective criterion ( Bapteste and Boucher 2009 ; Lawrence and Retchless 2009 ). The recent availability of genome sequence data now allows the application of the same parameters for delineating species boundaries to asexual lifeforms (bacteria, archaea, viruses), all of which were previously considered as not amenable to classification based on the BSC ( Donoghue 1985 ; Rosselló-Mora and Amann 2001 ; Costechareyre et al. 2009 ). This uniformity in defining species has implications beyond taxonomic classification in that the formation of equivalently defined species allows comparisons of evolutionary processes across all lifeforms ( Staley 2009 ) and more accurate inferences about the rates and patterns of speciation in different groups of organisms.

The ANI divergence between strains in E. coli BIO can be as much as 6.1%. This relatively high level of divergence between members of the same species is evident at other taxonomic ranks: for example, between E. coli BIO and other species of Escherichia ( E. albertii , E. fergusonii, and Clades II, III, IV, and V), sequence divergence ranges from 8% to 12%, and between Escherichia and its sister genus, Salmonella , the divergence among shared genes averages 15%. This degree of variation within and among species sharply contrasts the situation in, say, humans, in which the sequence divergence between homologs from two individuals is a mere 0.1% ( Lek et al. 2016 ), and there is only a 0.5% difference to our sister species Homo neanderthalensis ( Noonan et al. 2006 ) and 1.2% difference to our sister genus Pan ( Carroll 2003 ).

The genetic approach to bacterial identification and classification, which began in the 1960s ( Marmur et al. 1963 ), is more instructive than metabolic typing, which relies on a subjective set of diagnostic features (which themselves can originate by different means within and across species, and are often not discrete) ( Priest et al. 1993 ). Moreover, a genetic delineation of biological species divulges the actual extent of phenotypic variation that is present in a species. For example, E. coli is traditionally distinguished from S. enterica as being Lac-positive and Citrate-negative; however, many members of E. coli , including most Shigella and many pathogenic strains, are lactose nonfermenters, and citrate-positive strains of E. coli have been reported ( Ishiguro et al. 1978 ) and evolved ( Blount et al. 2012 ). All of the classification methods that we evaluated indicate that the majority of E. coli and Shigella represent a single species; however, our analyses, based on the propensity for homologous exchange, provide the genetic basis for this conclusion.

Reports that strains within some phylogenetic clades of E. coli recombine at higher frequencies within one another than with members of other clades—as might be expected if homologous exchange relied wholly on the degree of sequence similarity—has been interpreted as evidence of incipient speciation ( Didelot et al. 2012 ; Kang et al. 2021 ). However, applying the principles of the BSC, we established the genetic boundaries of E. coli , termed E. coli BIO , which was found to include all members of the genus Shigella , exclude only 12 genomes currently classified as E. coli in the NCBI database, and to be distinct from the other named species within the genus. Aside from its utility in classification and systematics, applying a universal species concept and identifying populations that readily engage in gene flow is valuable for studying novelty and diversity within species, and the mechanisms by which bacterial species form.

Materials and Methods

Genomes analyzed.

We downloaded a total of 1,635 genome sequences classified as E. coli by the NCBI database ( www.ncbi.nlm.nih.gov/ ), which included representatives of the species within E. coli recognized by the GTDB v207 (April 8, 2022; gtdb.ecogenomic.org/) ( Parks et al. 2018 ) and the E. coli phylogroups of Abram et al. (2021) . To maximize core-genome size, we restricted our analyses to all complete, ungapped genomes available at the time of analysis. Additionally, we retrieved complete genome sequences for Escherichia species other than E. coli ( E. albertii , n = 1; E. fergusonii , n = 2; and 53 Escherichia strains not assigned to species), the five Escherichia phylogroups (CI–CV) described by Walk et al. (2009) ( n = 12), the four named species of Shigella ( S. flexneri, n = 28; S. boydii, n = 9; S. dysenteriae, n = 5; S. sonnei, n = 34), one unassigned strain of Shigella , S. enterica ( n = 106), S. bongori ( n = 5), E. cloacae ( n = 3), Klebsiella pneumoniae ( n = 3), P. mirabilis ( n = 2), and one strain each of Citrobacter koseri , Citrobacter rodentium, Cronobacter sakazakii , Cronobacter turicensis, Enterobacter cancerogenus , Enterobacter lignolyticus, Enterobacter sp., and Klebsiella variicola . Accession numbers, strain, and species assignments and nomenclature in the NCBI and GTDB databases (and Walk et al. and Abram et al. phylogroups, where applicable), and taxonomic classification based on the schemes implemented in this study, are presented in supplementary table S1, Supplementary Material online.

Initial assignment of genomes to a named species followed the nomenclature designated in the NCBI database. Currently, the NCBI database uses an ANI metric to assign genomes to species, with species-level assignments representing strains having >95% ANI for at least 90% of the shared portions of their genomes ( Ciufo et al. 2018 ). Assignments to bacterial genera do not rely on fixed ANI cutoffs, and accommodations are made for certain genera, such as Shigella, which is known to be polyphyletic and contained within E. coli. The GTDB also defines species based on >95% ANI to a representative strain, except in cases in which representatives from different species, as obtained from cross-referencing the LPSN, BacDive, StrainInfo, and NCBI databases, are very closely related and a higher threshold must be applied.

Classification Methods and Detecting Gene Flow Among Strains

Complete genomes were partitioned into sets according to their nomenclature, phylogenetic groupings, or degree of DNA similarity. For each selected set, we evaluated the extent of recombination among genomes and the consistency among the taxonomic assignments based on different methods and criteria. We applied and compared the following methodologies for species-level classification:

• (i) Average Nucleotide Identity (ANI). We calculated ANI, a whole-genome metric for evaluating the degree of DNA sequence identity, using FastANI ( Jain et al. 2018 ). When assigning strains to E. coli by this approach, we applied the “one-to-many” option and used the type strain E. coli ATCC 11775 ( https://lpsn.dsmz.de/species/escherichia-coli ), which was fully sequenced in 2019 ( Wadley et al. 2019 ), as the species representative to which all other genomes were compared. As such, all genomes with an ANI ≥ 95% to ATCC 11775 would be designated members of E. coli . We also applied the “many-to-many” option in FastANI employing the same DNA identity threshold.
• (ii) ConSpeciFix . To identify species boundaries according to the precepts of the BSC, we used the ConSpeciFix v1.3.0 pipeline ( Bobay et al. 2018 ), which recognizes genomes as belonging to the same species based on their capacity for gene flow. In ConSpeciFix , gene flow is estimated by assessing the extent of homologous recombination among genes in the core genome. The core genome is built with single-copy orthologs that occur in at least 85% of all strains considered, with single-copy orthologs aligned in MAFFT v7 ( Katoh and Standley 2013 ) and merged into a single concatenate. Based on the core-genome phylogeny, ConSpeciFix calculates the number of homoplastic alleles ( h, recombinant sites, i.e., those not related by vertical ancestry) relative to the number of nonhomoplastic alleles ( m, vertically transmitted mutations), using a distance-based approach, with higher h/m ratios indicative of more recombination ( Bobay et al. 2018 ).

To calculate h/m ratios, which estimates the limits of recombination among genomes, a representative of a different species or phylogenetic clade (the “test lineage”) is included in a set of genomes previously determined by ConSpeciFix to recombine with one another (the “reference lineages”). Disruptions or reductions in h/m values caused by the inclusion of the test lineage indicate that the test lineage does not recombine with the reference lineages and, thus, belongs to a different species based on the BSC. Analyses were extended to include different combinations of reference genomes and test lineages in order to define species boundaries.

To define the set of E. coli genomes that constitute the reference lineages, we initially examined the 1,635 complete genomes available in the NCBI database. Because it was computationally infeasible to run the entire set of genomes through the ConSpeciFix pipeline as a single group, we randomly subdivided those strains designated as E. coli into subgroups of 150 genomes and analyzed each subgroup separately. These analyses identified 12 genomes that were reproductively isolated from the rest of the E. coli genomes and, therefore, removed from the set of NCBI-designated E. coli strains that were randomly sampled to produce new sets of reference lineages for assessing recombination with test lineages. Within the ConSpeciFix pipeline, we also tested the extent of gene flow between E. coli and representative genomes of other species of Escherichia , the four species of the genus Shigella, and several non- Escherichia species of Enterobacteriaceae ( supplementary table S1, Supplementary Material online).

• (iii) PopCOGenT . Another approach for defining bacterial species based on gene exchange, PopCOGenT ( Arevalo et al. 2019 ), uses the presence of anomalously similar regions to infer events of gene transfer between genomes. Unlike ConSpeciFix , which deduces the source and ancestry of each polymorphic site, PopCOGenT is based on the premise that SNPs occur more frequently in vertically inherited genes than in recently transferred regions, and that genomes engaging in gene exchange will have longer and more frequent stretches of identical regions. In both ConSpeciFix and PopCOGenT , genomes connected through gene exchange are considered members of the same species, but the methods differ in criteria for identifying recombination, and possibly, species boundaries.

We applied PopCOGenT to a total of 128 genomes, including many that were originally assigned to E. coli but whose species status has been questioned or changed. In addition to strains consistently classified as E. coli , this set included strains labeled as E. ruysiae and E. marmotae by the GTDB, the 12 E. coli genomes from the NCBI database recognized by ConSpeciFix as being reproductively isolated from the rest of the species, representatives of the CI–CV phylogroups ( Walk et al. 2009 ) as well as representatives of other species ( E. albertii , E. fergusonii , Shigella sp. PAMC 28760, Shigella sonnei , S. flexneri, S. dysenteriae, Shigella boydii , S. enterica, and S. bongori ) and the phylogroups of Abram et al. (2021) ( supplementary table S1, Supplementary Material online).

To compare the species assignments and nomenclature of Escherichia and Shigella strains across different classification schemes, we first dereplicated the dataset, retaining only a single representative strain for cases in which multiple genomes averaged >99.8% nucleotide identity. This dereplication yielded a reduced, but otherwise identical, phylogeny that was used for ConSpeciFix . The maximum-likelihood phylogeny of the 70 genomes remaining after dereplication was generated with RaxML ( Stamatakis, 2014 ) using the analysis tool PhaME ( Shakya et al. 2020 ). To generate this phylogeny, PhaME uses nucmer2 ( Delcher et al., 2002 ) to first aligns each genome against itself in order to identify and eliminate the repeated regions within a genome, and then aligns the repeat-free genomes against the selected reference genome. The RaxML phylogeny of the aligned genomes with associated bootstrap branch-support values was built using the evolutionary model GTR and the rate heterogeneity model GAMMA with an estimation of invariable sites (GTRGAMMAI).

Supplementary Material

Msac273_supplementary_data, acknowledgments.

We thank Kim Hammond for assistance with figures. This work was supported by the National Science Foundation Dimensions (grant number 1831730) and the National Institutes of Health (R35GM118038) to H.O.

Contributor Information

Marta Cobo-Simón, Department of Molecular Biosciences, University of Texas at Austin, Austin, TX.

Rowan Hart, Department of Molecular Biosciences, University of Texas at Austin, Austin, TX.

Howard Ochman, Department of Molecular Biosciences, University of Texas at Austin, Austin, TX.

Supplementary data are available at Molecular Biology and Evolution online.

Author Contributions

H.O. conceived the study; H.O. and M.C.S. supervised the research activity planning and execution; M.C.S. and R.H. analyzed and interpreted the data; M.C.S., R.H., and H.O. wrote the paper, read, and approved the final manuscript.

Data Availability Statement

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