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Review

From Cryptic Clade to Emerging Pathogen: Exploring the Evolutionary Divergence and Clinical Relevance of Escherichia marmotae

by
Pelumi Oladipo
1,2,*,
Ayomikun Kade
3,
Hope Onohuean
4,5 and
Jeffrey L. Ram
1,*
1
Department of Physiology, School of Medicine, Wayne State University, Detroit, MI 48201, USA
2
Department of Biochemistry, Microbiology, and Immunology, School of Medicine, Wayne State University, Detroit, MI 48201, USA
3
School of Life Sciences, Gibbet Hill, University of Warwick, Coventry CV4 7AL, UK
4
Biomolecules, Metagenomics, Endocrine and Tropical Disease Research Group (BMETDREG), Kampala International University, Western Campus, Ishaka-Bushenyi P.O. Box 20000, Uganda
5
Biopharmaceutics Unit, Department of Pharmacology and Toxicology, School of Pharmacy, Kampala International University, Ishaka-Bushenyi P.O. Box 20000, Uganda
*
Authors to whom correspondence should be addressed.
Microorganisms 2026, 14(4), 869; https://doi.org/10.3390/microorganisms14040869
Submission received: 24 February 2026 / Revised: 31 March 2026 / Accepted: 7 April 2026 / Published: 13 April 2026
(This article belongs to the Special Issue The Microbial Pathogenesis)
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Abstract

The Escherichia genus includes both commensal and pathogenic species and is characterized by its diversity and adaptability to the mammalian gut and other environments. Among these species, E. coli has facilitated many scientific advances as a model organism. Recently, a new member of the Escherichia genus, Escherichia marmotae, has been described as a phylogenetically distinct clade that shows the greatest genetic divergence from E. coli. This review explores E. marmotae, its cryptic evolution, distinct characteristics, and ecological niches. E. marmotae has recently gained scientific prominence due to its association with animal feces, environmental occurrence, human clinical samples, and emerging as a potential pathogen. While its pathogenicity remains understudied, growing evidence from clinical, environmental, and animal sources suggests the need for heightened surveillance. This review highlights current knowledge gaps, underscores the need for improved diagnostic tools, and proposes future research directions to elucidate the clinical and ecological implications of this emerging pathogen.

Graphical Abstract

1. Introduction

The genus Escherichia currently comprises six recognized species: Escherichia coli [1], Escherichia fergusonii [2], Escherichia albertii [3], Escherichia ruysiae [4], Escherichia whittamii [5], and Escherichia marmotae [6]. Among these, E.coli remains the most extensively studied; however, increasing attention has recently been directed toward other Escherichia species with emerging clinical relevance, including E. marmotae.
Escherichia marmotae was initially described as cryptic clade V, a phenotypically identical but genetically distant relative of Escherichia coli. However, in 2015, scientists isolated this cryptic clade from the feces of Marmota himalaya in Tibet, characterized its phenotypic properties, and named it Escherichia marmotae [6]. Since its initial isolation from Himalayan marmot feces and environmental waters [6,7], the organism has also been found in the diaphragms of wild boars [8], and the feces of cows, bank voles [9,10], clams [11], arctic fox [12] rodent [13], farm healthy hens [14], and birds [15,16,17,18]. E. marmotae has recently gained additional scientific prominence due to its association with human infections and genotypic virulence traits associated with Escherichia pathogenicity [19,20,21,22].
In 2022, E. marmotae was first identified in clinical samples by genome sequencing of isolates from cases of thoracic spondylodiscitis, pyelonephritis, acute sepsis of unknown origin, and postoperative sepsis [19]. The infective organisms were initially misidentified as E. coli using Matrix-Assisted Laser Desorption Ionization—Time of Flight Mass Spectrometry (MALDI-TOF MS), but with slightly divergent spectral profiles and low confidence scores [19]. Subsequent sequencing of their 16S rRNA amplicon unambiguously identified the organisms as E. marmotae, and this was later confirmed by whole-genome sequencing. Similarly, Sinha et al. (2023) identified a UTI-associated strain of Escherichia as E. marmotae [20]. The first clinical strain identification in North America was from a patient with sepsis in Detroit, MI, US [22]. The increased identification of E. marmotae in human infectious diseases, together with diagnostic challenges, necessitates the need for a comprehensive evaluation of this emerging pathogen. This review aims to contextualize the emergence of E. marmotae within the broader framework of Escherichia evolution. We explore the distinguishing genomic features of E. marmotae, its ecological reservoirs, and the mounting clinical evidence suggesting its role as a potential human pathogen. Additionally, we highlight the diagnostic challenges associated with its identification, discuss its antimicrobial resistance profiles, and provide recommendations for future research to better define its clinical and public health significance.

1.1. Escherichia coli and Related Species

Escherichia coli, a Gram-negative bacterium, is a well-known commensal resident primarily in the lower intestinal tracts of humans and warm-blooded animals, as well as water, sediment, and soil [1]. Its presence in water is often indicative of fecal contamination from warm-blooded animals [23]. Although most commensal strains of E. coli are relatively harmless, pathogenic strains can cause intestinal and extraintestinal diseases due to its virulence factors [24]. Its pathogenic roles span from intestinal infections, urinary tract infections (UTIs), and hospital-acquired pneumonia (HAP) to more severe conditions such as hemolytic-uremic syndrome (HUS), meningitis, and septicemia.
The Escherichia genus also includes other traditionally distinct species such as Escherichia fergusonii. E. fergusonii is genetically the most similar to E. coli, sharing approximately 64% DNA-DNA hybridization, which makes differentiating with 16s rRNA gene sequencing alone challenging [2,7,25]. Less similar species, such as E. albertii is also a member of the genus [7,26]. Traditionally, biochemical tests can be used to distinguish E. coli from E. fergusonii by its ability to ferment both lactose and sorbitol, except for certain pathogenic E. coli strains such as O157:H7, which do not ferment sorbitol [2,27,28].
Additionally, non-toxin producing strains of E. coli express the β-glucuronidase gene (uidA), and produce β-glucuronidase enzyme, which can be detected by a variety of different assays, including IDEXX’s Colilert, which contains two nutrient indicators, ONPG (ortho-nitrophenyl- ß-D-galactosidase) and MUG (4-methyl-umbelliferyl- ß-D- glucuronide) [29]. However, phylogenetic analysis based on housekeeping genes provides the most effective method of distinguishing E. coli from E. fergusonii and E. albertii, allowing for precise delineation between these closely related species [27,30].

1.2. Cryptic Clades Genomic Divergence and Ecological Adaptation

Despite the intensive study of E. coli as a model organism over a century, the discovery in the 21st century of strains that are indistinguishable phenotypically from E. coli but genetically divergent was particularly surprising [7]. Cryptic clades within the Escherichia genus were discovered through population-genetic analysis of the uidA gene in E. coli isolates from surface water, sewers, and the feces of humans, birds, raccoons, and dogs [31]. Multilocus sequence analysis based on 22 core genome genes revealed that these strains fell into five novel ‘cryptic’ lineages [7]. Evolutionary analysis using a minimum evolution tree to estimate divergence suggests that E. albertii, E. fergusonii, Clade II, and Clade V split between 38 and 75 million years ago, while E.coli, Clade I, Clade III, and Clade IV 19 diverged 31 million years ago [7].
In 2018, Cryptic Clade VI was added to the cryptic clades [32,33,34]. Recently, Mire et al. [35], reported the addition of three cryptic clades (Clade VI–Clade VIII), expanding the number to eight clades. However, the majority of available studies have focused on cryptic clades I–V, while clades VI–VIII remain mostly uncharacterized. Over the years, several cryptic clades have been renamed, such as cryptic Clade II and Clade V, which are now known as Escherichia whittamii and Escherichia marmotae respectively, while Escherichia ruysiae sp. encompasses the closely related Escherichia Cryptic Clades III and IV (Figure 1, Table S1). Pairwise comparison of the full genomes of isolates of E. marmotae to representative sequences of E. coli indicated that these genomes differed on average by 9.4% over all genes [36]. Genetic distances from E. coli to various cryptic clades estimated from single-nucleotide polymorphisms (SNPs) of core genome genes averaged 3.2% for Clade I, 7.5% for Clade II, 6.7% for Clade III, 6.5% for Clade IV, and 8.1% for Clade V, emphasizing the greater divergence of Clade V (E. marmotae) from E. coli in comparison to the other cryptic clades [37].
The cryptic clades inhabit ecological niches different from those of E. coli. Clades II–V are relatively abundant and well-suited to non-intestinal environments, such as freshwater beaches and surface water [33]; whereas, Cryptic Clade I favors the intestinal environments where they appear to provide functions that enhance environmental survival, such as diol utilization and lysozyme production [38,39]. Cryptic clades have also been identified in other animal hosts, particularly birds [16,40]. The cryptic clades have been shown to have the potential to cause extraintestinal infections through the expression of distinct virulence factors. Interestingly, Ingle et al. [41] revealed that Cryptic Clades III, IV, and V form biofilms better than the E. coli, E. albertii, and E. fergusonii strains. They can replicate at temperatures as low as 5 °C, unlike E. coli and E. fergusonii, which usually require at least 11 °C. While a mouse model of extraintestinal infection shows they are non-virulent, their ability to survive in harsh environments, coupled with their robust biofilm formation, raises concerns about their potential as opportunistic pathogens [41].

2. Molecular Detection and Pathogenic Potential of Cryptic Clades

Historically, identifying and differentiating non-E. coli from E. coli strains [7] relied on sequencing specific genes, which is time-consuming and labor-intensive. However, in 2011, Clermont et al. developed a PCR-based technique to distinguish E. coli from various cryptic clades of Escherichia [40]. The approach involved allele-specific PCR reactions targeting the chuA, outer membrane heme, and aes (acetyl esterase B) genes with clade-specific primers, resulting in PCR products of distinct sizes, unique to each clade. The method proved effective even when traditional tests failed to differentiate them. This coincidentally identified the first suspected instances of cryptic clades (Clades III and V) associated with human infections, since these isolates were from a large collection of Escherichia isolates from septicemic patients in France [42].
A study by Vignaroli et al. [39] identified 20 cryptic clades among 138 E. coli strains isolated from marine sediments, using the aes and chuA allele-specific primers developed by Clermont et al. [40] to investigate the pathogenic potential of the cryptic clades. The study assessed the adhesion and invasion ability of the cryptic clades using Caco-2 and Int-407 mammalian intestinal cell lines. Among the cryptic clades, Cryptic Clade V had the highest adhesion index, comparable to the positive control E. coli ATCC35150 [39]. However, none of the cryptic clades could invade intestinal cell lines. This lack of invasive ability of Cryptic Clade V on epithelial cells was also observed in a study by Zhurilov et al. [9]. In contrast, in 2019 Liu et al. [43] demonstrated that E. marmotae possesses some invasive capacity, albeit with a lower invasion index than Shigella, a known invasive pathogen. These results indicate that various E. marmotae strains may differ in their invasive capability and thereby in their potential to act as invasive pathogens.
Vital et al. [44] investigated the gene expression profiles of Escherichia Cryptic clades under different environmental conditions. Escherichia Cryptic clades exhibit high expression levels of stress defense genes under starvation conditions, upregulating genes associated with acid stress defense, desiccation stress defense, and toxic compound stress defense, thus enhancing the capacity for survival [44]. These adaptations ensure survival and resilience in adverse conditions. Cryptic clades isolated from marine sediments [45] were studied for biofilm formation, resistance to hydrogen peroxide, and expression of the “red, dry, and rough” (rdar) morphotype, a phenotype characterized by the production of extracellular matrix composed of curli fimbriae and cellulose [46]. Out of 28 cryptic clade isolates tested, 79% were weak or non-producers of biofilm at 37 °C and lacked the surrounding extracellular matrix. However, growth at 24 °C led to significant biofilm formation and expression of the rdar morphotype [45]. The co-expression of curli and cellulose plays an important role in biofilm formation, cell aggregation, and cell adhesion [45,47]. Future work to characterize how genomic variations among the cryptic clades cause phenotypic changes are potentially important for determining their pathogenicity.
Although initial studies suggested that E. whittamii, E. ruysiae, and E. marmotae were not significant public health risks [38], recent reports of E. marmotae, associated with human infections [19,20] indicate a potential pathogenic role for E. marmotae. Further investigation is necessary to fully understand its pathogenic potential.

3. Cryptic Clades as Indicators of Fecal Contamination in Water Quality Monitoring

Fecal contamination of recreational waters is a significant source of pathogens that cause waterborne diseases. E. coli has been the gold standard indicator of fecal contamination [48]. Conventional testing methods enumerate E. coli by detecting the expression of the uidA gene, which encodes beta-D-glucuronidase enzyme [29,30]. Detection of beta-glucuronidase-positive colonies and cultures is recommended by the US Environmental Protection Agency (USEPA) for monitoring fecal pollution in water (USEPA, 2002). However, other species within the genus Escherichia also possess this gene, potentially leading to confounding interpretations of water quality, as environmental bacteria do not indicate the presence of harmful fecal material.
Environmentally adapted “naturalized E. coli” may contribute to an increased number of E. coli counts and lead to false alarms of fecal pollution. Several studies have suggested detecting “cryptic clades” in the current E. coli-based water quality monitoring methods might overestimate the health risk of faecal contamination [7,40,49]. To address this issue, Mire et al. [35], developed a specific PCR assay targeting a gene called ecc (Escherichia Cryptic clades), which is specific to only cryptic clades as confirmed by sequencing their whole genomes. Deng et al. [50] also proposed that the ycjM gene, a putative glycosyltransferase gene, is a distinctive genetic marker to differentiate enteric E. coli from naturalized E. coli. The study hypothesised that ycjM is present in host intestinal tracts and absent in environmental E. coli and non-E. coli bacteria [50]. Genetic techniques have been developed to discriminate enteric–fecal indicator bacteria from naturalized E. coli by the presence of enteric-associated genes for gut colonization and several prophage genes [38,51].
Ecological analysis has shown that members of Clade I are often isolated from human and animal feces [41,52], indicating their potential as fecal indicator bacteria [35]. In contrast, isolates of clades II through V are mostly collected from water or soil samples [7,33,39,40]. E. marmotae and E. ruysiae have been isolated from environmental, animal, and human sources, and their genotypic characteristics are similar to those of Cryptic Clade I, presenting a compelling case for their inclusion as fecal indicators alongside Cryptic Clade I [19,53]. The variation in these clades across environments, coupled with the inconsistent presence of the ycjM gene among strains, underscore that they may be identified as indicators of fecal pollution.

4. Cryptic Clades

4.1. Cryptic Clade I

Cryptic Clade I exhibits the characteristic virulence traits of E. coli, including their potential to cause human infections [40,41,52]. Thus, E. coli and Escherichia Clade I are now designated as E. coli sensu lato, and the classic E. coli (designated phylogroups A–G) are referred to as E. coli sensu stricto, which are genetically closer to E. coli than other clades [54]. Cryptic Clade I are predominantly enteric, commonly found in animal gastrointestinal tracts, and less frequently in external environments [7,38,41]. These strains have been implicated in human infections, including those isolated from children with diarrhea.
Significant findings indicate that clade I strains capable of producing ETEC enterotoxins, as well as STEC strains, have been isolated from human patients [33,52] and from animals [32,33,55]. Distinguishing clade I from E. coli is important because Clade I strains harbour virulence factors that cause human diarrhea. Okuno et al. [56] developed a PCR-based method to distinguish Clade I from E. coli to help avoid false identification of cryptic clades. The method relies specifically on a gene of unknown function, 1368 bp in length, that contains two secreted effector protein pipB2 domains, which are conserved in clade I strains and absent in other members of the Enterobacteriaceae [56].

4.2. Escherichia whittamii

Cryptic clade II was named Escherichia whittamii in honor of the American microbiologist Thomas S. Whittam [5]. Whittam made significant contributions to microbiology, particularly in the collection and characterization of E. coli strains and the identification of the cryptic clades. Although E. whittamii shares its genus with the well-known E. coli, it remains under-researched and is considered rare [40]. It has been isolated from diverse biological sources, including bird feces [7], chicken gut [5,14], and human fecal samples [40], indicating its widespread presence. However, the ecological niche and characteristics of E. whittamii need further exploration.
Recent studies by Shen et al. [57,58] investigated the genetic diversity and potential adaptation of strains of E. whittamii isolated from intertidal sediments. Through comprehensive genomic analysis, they revealed ecological differentiation within this cryptic clade. Delta bitscore metrics, a method for quantifying sequence alignments, were used to assess evolutionary divergence within E. whittamii [59]. The study identified unique genetic signatures indicative of Escherichia whittamii’s adaptation to distinct ecological niches.

4.3. Escherichia ruysiae

Escherichia ruysiae, formerly classified within cryptic clades III and IV, has been predominantly isolated from environmental samples [7]. However, its detection in a range of animal and human-associated sources indicates a wider host range than previously recognized [60]. E. ruysiae was first isolated from a fecal sample of an international traveler with diarrhea [4], demonstrating its ability to colonize the human gastrointestinal tract. More recently, a clinical isolate was recovered from a five-year-old female patient hospitalized with gastroenteritis and bloody diarrhea, further suggesting its potential relevance in human disease [61].
Beyond human-associated cases, E. ruysiae has been detected in multiple animal reservoirs. Isolates have been recovered from a Lohmann Brown layer hen (Gallus gallus domesticus) on a farm in England [14] and wild crow in Japan [18]. Its presence in the fecal microbiome of a healthy domestic dog in the United States highlights its ability to colonize companion animals and supports its broad geographic distribution [60]. Collectively, these findings suggest that E. ruysiae is geographically widespread, with reports spanning North America, Europe, Asia, and Africa [4,8,18,60,61,62].
Despite its detection across diverse hosts and environments, the ecological distribution, transmission pathways, and potential role of E. ruysiae in human and animal health remain poorly characterized. Genomic analyses have identified several virulence- associated genes, such as those involved in siderophore function (chuX, entS, fepABD), fimbriae (fimBCDGI), heat-stable enterotoxins (astA), adhesion and biofilm formation genes (csgABCDEFG and agn43), chemotaxis regulatory protein (CheRBYZ), ompT gene for outer membrane protease, flagellar protein (flgABCDE, fliABC, flhABC, and motAB), fitness (iutA and kpsMII), type II secretion systems (gspGHI), and capsular polysaccharide biosynthesis (kpsD), none of which are strictly associated with a defined E.coli pathotype. Some of them can be found in nonpathogenic E.coli [8,18,61,62,63].
A smaller subset of E. ruysiae isolates harbor virulence determinants more commonly associated with pathogenic E. coli, including dispersin (aap/aspU), the aat secretion system (aatABCP), enterotoxin gene senB, colicin Ib (cib), plasmid R100 complement resistance gene (traJT), all of which may contribute to diarrheal infections [18,61,62]. Additionally, the presence of the sat gene typically associated with uropathogenic E. coli (UPEC) and enteroaggregative E. coli (EAEC), further suggests the potential for pathogenic behavior [63]. However, no consistent association between E. ruysiae and specific clinical syndromes, including diarrheal disease, has been established, complicating efforts to define its pathogenic capabilities [4,11,62].
Escherichia ruysiae harbors a range of antimicrobial resistance genes (ARGs), including extended-spectrum β-lactamases (ESBLs) such as blaCTX-M-15, blaCTX-M-14, and blaAmpC, variants, indicating resistance to β-lactam antibiotics [4,60,61,62]. Additional resistance determinants include genes associated with aminoglycosides (aadA, aph) [4,60,62], fluoroquinolones (qnrS1) [62], trimethoprim (dfrA) [62], sulfonamides (sul2) [4,62], and tetracycline (tet(A) [62]), as well as multidrug efflux systems (emr, edt) [61].
Accurate identification of E. ruysiae presents significant challenges in clinical laboratory settings. Conventional biochemical identification methods and automated systems such as VITEK2 and MALDI-TOF mass spectrometry frequently misidentify E. ruysiae as E. coli due to highly similar phenotypic characteristics [4,18,60,61,62]. At present, E. ruysiae is not considered a human pathogen, and its role as a zoonotic pathogen in human populations remains unclear. Further studies and enhanced surveillance are needed to understand its ecological roles and resistance mechanisms and potential contribution to human disease.

4.4. Escherichia marmotae

E. marmotae has been found in various environments, inhabiting the diaphragms of wild boars, the rectal feces of cows [10], bank voles [9], farm healthy hens [5], clams [11], birds [17,18], and wild boars [8]. The prevalence of human infection caused by E. marmotae remains unclear [18,19,20,22].
This broad host range suggests that E. marmotae may represent an emerging pathogen that has been overlooked amidst the many invasive E. coli strains processed in clinical microbiology laboratories. Historically, MALDI-TOF MS could not reliably distinguish E. marmotae from E. coli [18,19,21,64,65]; however, detection has recently improved with the identification of a discriminatory spectral peak at 7260–7268 m/z [22]. Numerous human infections associated with E. marmotae have now been documented (Table 1), with most cases initially misidentified as E. coli.

4.4.1. Phenotypic Characterization and Physiology of Escherichia marmotae

E. marmotae is a Gram-negative, facultatively anaerobic rod [6]. Phenotypically, it closely resembles Escherichia coli, which contributes to its frequent misidentification in routine clinical microbiology laboratories. E. marmotae exhibits an Escherichia-like biochemical profile; catalase-positive, oxidase-negative, hydrogen sulfide (H2S) negative, and urease-negative [6,9,21]. However, strain-level variability has been reported in several biochemical reactions, including indole production, β-galactosidase (ONPG) activity, lactose fermentation, ornithine decarboxylase activity, and carbohydrate utilization profiles. This variability likely contributes to its frequent misidentification as atypical or inactive E. coli.
Among the Escherichia cryptic clades, E. marmotae is the most studied, but many areas still need investigation. Contradictory reports about the motility of E. marmotae have been published, with some identifying it as motile at human body temperature [9] and others as non-motile [6,13,22,64]. A recent study showed that E. marmotae is motile and possesses flagella at 28 °C but not at 37 °C (Figure 2) [36]. Further investigation is needed as motility could significantly impact the infection process. Temperature-dependent changes in motility have been observed in E. marmotae and require further investigation, especially regarding the genes that mediate these behaviors [36].
Similarly, biofilm formation in E. marmotae appears to be influenced by temperature [36]. Studies have shown that they produce more robust biofilms at lower temperatures [36,41], than at 37 °C, consistent with increased expression of extracellular matrix components, such as curli fimbriae and cellulose. At host temperature, biofilm formation is reduced, and extracellular matrix production may be diminished despite the presence of underlying genetic determinants, as reported for cryptic Escherichia clades [45]. This indicates that environmental cues play a significant role in regulating surface-associated phenotypes.

4.4.2. Genomic Features, Virulence, and Antibiotic Resistance

The genome size of E. marmotae typically ranges from 4.2 to 5.2 Mbp, making it slightly smaller on average than E.coli [6,10,13,36,53,64]. As noted above in Section 1.2, the average nucleotide identity (ANI) based on whole-genome sequences shows about 9% similarity between E. marmotae and E. coli and other Cryptic clades, with intra-species similarity >98% [36,66]. Based on 16S rRNA gene sequences, E. marmotae exhibits >99.9% identity to other E. marmotae strains and approximately 99.2% similarity to E. coli [6,9,13,20]. Comparative genomic analyses further show that E. marmotae strains share more than 3000 core genes and a large accessory genome [19,36].
Various studies have identified numerous virulence genes within E. marmotae in environmental and clinical strains [6,17,19,20,21,22,36,64]. These include the enterotoxin encoding gene astA, iron acquisition systems (entA/B/C/D/E/F/S), heme uptake (fecA/B/C/DE) and metabolism (chuS/T/U/V/W/X), and genes involved in the production of curli proteins (csgB/D/E/F/G) and type 1 fimbriae (fimA/B/C/D/E/F/G/H/I), adherence, regulatory factors (gadX, H-NS, marA), bacterial secretion systems (gspC/D/E/F/G/H/I/J/K/L/M), and invasion factors (lbeA/B/C) [6,8,9,19,20]. These genetic components aid bacterial survival during invasive infections by promoting cellular adhesion, biofilm formation, and increased virulence. Most strains of E. marmotae possess outer membrane protein A, which has been associated with pathogenic properties that enable bacteria to breach the blood–brain barrier and evade host defense mechanisms, as observed in Neonatal meningitis E. coli (NMEC) [67].
Experimental studies suggest that virulence in E. marmotae may be strain-dependent. A recent study by Zhurilov et al. [9] examined a single E. marmotae strain (M-12) isolated from the lungs of a wild bank vole. When administered to mice, M-12 did not exhibit measurable virulence, consistent with earlier observations by Ingle et al. [41]. Despite this, the strain showed strong adhesion to HEp-2 cells and red blood cells, likely due to curli and fimbrial genes. Although biofilm genes were present in the genome, M-12 did not form biofilms at 37 °C [9], consistent with reports that E. marmotae exhibits enhanced biofilm formation at lower temperatures [36,41].
Plasmid-associated virulence has also been reported. Among the strains of E. marmotae, a unique isolate from the feces of the Himalayan marmot (Marmota himalayana) has been identified with a type III secretion system (T3SS) and other virulence genes found within the pEM148 plasmid. This plasmid of E. marmotae HT073016 [6] exhibits a genetic similarity ranging from 45% to 94% with a large virulence plasmid pCP301 (pINV) of Shigella flexneri, with almost the entire gene cluster but lacking the virF gene, a master activator needed for expression of the invasive phenotype [68]. Cell invasion experiments also showed that this strain is less invasive than Shigella, an invasive pathogen [43].
Genetic variability has been observed for mobile genetic elements (MGE), plasmids, and prophages, supporting the frequent gene gain and loss across the species [64]. Plasmid analyses reveal the presence of multiple incompatibility (Inc) groups, including IncF, IncI, and IncN, many of which are associated with conjugative transfer. Notably, E. marmotae has been shown to acquire clinically relevant resistance plasmids, such as IncL plasmids carrying blaOXA-48 [65] as well as AmpC- and ESBL-associated genes shared with co-existing E. coli populations [17]. Antimicrobial resistance genes are frequently linked to transposable elements, highlighting the role of MGEs in facilitating gene acquisition and dissemination. Collectively, these findings indicate that E. marmotae harbors a dynamic mobilome that supports adaptation across diverse ecological niches and contributes to the spread of antimicrobial resistance.
Reports of antibiotic resistance in E. marmotae have increased in recent years [11,17,21]. A multidrug-resistant (MDR) E. marmotae strain was reported from a 64-year-old man hospitalized with febrile neutropenia—the only documented MDR case in the Czech Republic [21]. Resistance to several antibiotics has been reported in both environmental and clinical isolates, including ampicillin [9,21], amoxiclav [9], tobramycin [9], tetracycline [17,36], ceftazidime [21], cefotaxime [21], cefazolin [36], erythromycin [13,36], and streptomycin [17]. Thus, E. marmotae has the capacity to acquire antimicrobial resistance, possibly through horizontal gene transfer and plasmid-mediated mechanisms. The apparent underreporting of resistant E. marmotae may be partly attributable to its frequent misidentification as Escherichia coli in routine clinical diagnostics, which could obscure its true contribution to antimicrobial resistance surveillance.
An E. marmotae strain isolated from clams was identified as an ESBL (Extended-Spectrum Beta-Lactamase) producer, showed phenotypic resistance to cefotaxime, and harbored the blaCTXM-1 gene [11]. Strains of E. marmotae isolated from a patient with a urinary tract infection contained blaKPC, blaCTX-M, and blaTEM-1b [20], highlighting the clinical relevance of E. marmotae in infections involving antimicrobial resistance.
E. marmotae exhibits variations in O antigens, and all E. marmotae have been shown to contain the fliC-H56 flagella antigen in both human and environmental strains [18,19]. E. marmotae may be truly pathogenic and able to cause severe infections. E. marmotae isolated from cow rumen was capable of potential biohydrogen production from sugarcane molasses under dark fermentation conditions [69]. E. marmotae has been reported to produce uricase by forming halo zones on uric acid agar plates while investigating potential uricase-producing strains from canal water near a dairy [70]. Uricase is an enzyme that facilitates the oxidation of uric acid into allantoin in the presence of oxygen. The enzyme has been used as a treatment to reduce the buildup of toxic urate, thereby serving as a therapeutic approach for managing gout and neuropathy [71].
Evidence points towards E. marmotae sharing similar pathogenic traits to extraintestinal pathogenic E. coli (ExPEC) and Enteroaggregative E. coli (EAEC). These traits are partly mediated by gene aaic and other gene components of the type VI secretion system [18]. ExPEC includes strains such as avian pathogenic E. coli (APEC), which share significant genetic similarities with human pathogenic strains, suggesting a potential zoonotic transfer of virulence and resistance genes [72].
The prevalence of illnesses caused by E. marmotae remains largely unknown, as does its role as a potential commensal organism in humans and animals. Given its shared genetic and phenotypic characteristics with pathogenic E. coli and the lack of a robust clinical test for E. marmotae, it seems likely that its occurrence is underreported. Another area that has hardly been explored is the influence of environmental factors on the expression of genes involved in virulence. These insights into the genomic and phenotypic variability of E. marmotae emphasize the necessity for research to understand better its clinical prevalence, pathogenic potential, and ecological roles.

5. Future Directions for Research Engagement

The classification of E. marmotae as either a human or an opportunistic pathogen remains an open question that requires further clarification. While some evidence suggests that E. marmotae has pathogenic potential, its role as a commensal organism in various environments and hosts complicates its categorization. Future research should focus on identifying the specific conditions under which E. marmotae transitions from a commensal to a pathogenic role. This includes investigating host-specific factors that may predispose individuals to infection, as well as environmental triggers that could influence its virulence. The exploration of genetic, ecological, and environmental factors influencing E. marmotae’s pathogenic potential will be critical in refining its classification and understanding its true clinical significance.
Improved clinical diagnostic methods for identifying the various cryptic clades are needed. MALDI-TOF MS has the potential to provide robust routine identification; however, several MALDI databases, including VITEK MS Prime by bioMérieux, lack spectra for E. marmotae. Bruker MALDI-TOF-MS includes E. marmotae in its Research Use Only (RUO) database, but independent documentation of its reliability is lacking. Updating these databases to include spectra for E. marmotae, E. ruysiae, and E. whittamii is critical to properly diagnosing Escherichia infections. Extensive research should focus on identifying unique peaks that distinguish these species from closely related pathogens, enhancing the diagnostic capabilities of MALDI-TOF MS. This technique has been used successfully to differentiate closely related bacterial species [73,74,75] and would significantly enhance the diagnostic capability of MALDI-TOF MS and support the early detection of infections caused by these organisms.
Comprehensive genomic studies are essential to uncover functional and genetic distinctions among E. coli, E. marmotae, and other Escherichia species. Understanding these differences will be crucial to assessing the pathogenic potential of the various Escherichia species, especially the roles of E. marmotae and the other formerly cryptic clades as potential zoonotic pathogens. Further research into how temperature-dependent conditions affect motility, biofilm formation, and virulence gene expression in E. marmotae will provide deeper insights into its environmental adaptability and potential pathogenic behavior in warm-blooded animals.

6. Conclusions

The study of E. marmotae has revealed its potential role as a human pathogen, harboring virulence factors, and being associated with various infections. Including these species in water quality monitoring will enhance the detection of fecal contamination and potential public health risks. Given its 91% genetic similarity to E. coli, there is a need for improved diagnostics. Investigating these genes, particularly those related to motility and temperature-dependent behavior, will provide insights into the adaptations and pathogenic mechanisms of these formerly “cryptic clades” of Escherichia.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms14040869/s1, Table S1: Strains of Escherichia coli and cryptic clades used for phylogenetic tree.

Author Contributions

Conceptualization, P.O. and A.K.; formal analysis, P.O.; investigation, P.O.; resources, P.O., and J.L.R.; data curation, P.O.; writing—original draft preparation, P.O., A.K., and H.O.; writing—review and editing, P.O., A.K., H.O. and J.L.R.; visualization, P.O. and A.K.; supervision, J.L.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ONPGOrtho-nitrophenyl-ß-D-galactosidase
UTIUrinary tract infections
MUG4-methyl-umbelliferyl-ß-D-glucuronide
HUSHemolytic-uremic syndrome
HAPHospital-acquired pneumonia
MALDI-TOF MSMatrix-Assisted Laser Desorption Ionization—Time of Flight Mass Spectrometry
ESBLsExtended-Spectrum β-lactamases
T3SSType III secretion system
RUOResearch use only
ANIAverage Nucleotide Identity
ExPECExtraintestinal Pathogenic E. coli
EAECEnteroaggregative E. coli
APECAvian Pathogenic E. coli
USEPAUS Environmental Protection Agency

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Figure 1. Grape tree showing the phylogenetic relationships among Escherichia coli and its cryptic clades.
Figure 1. Grape tree showing the phylogenetic relationships among Escherichia coli and its cryptic clades.
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Figure 2. Transmission electron micrographs of E. marmotae 3032. The panels illustrate temperature-dependent flagellar expression, with flagella present after incubation at 28 °C (right panel) and absent/reduced at 37 °C (left panel). Image adapted from Oladipo et al. 2025 [36].
Figure 2. Transmission electron micrographs of E. marmotae 3032. The panels illustrate temperature-dependent flagellar expression, with flagella present after incubation at 28 °C (right panel) and absent/reduced at 37 °C (left panel). Image adapted from Oladipo et al. 2025 [36].
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Table 1. Clinical cases of E. marmotae associated with human infections.
Table 1. Clinical cases of E. marmotae associated with human infections.
Source of InfectionDiagnostic MethodsTreatmentCountryReferences
80-year-old male with acute mylgenous leukaemia and pancytopenia diagnosed with thoracic spondylodiscitis
  • MALDI-TOF misidentified it as E. coli
  • 16S rRNA sequencing
Expanded-spectrum cephalosporin followed by a course of oral ciprofloxacin.Norway[19]
Pyelonephritis
  • MALDI-TOF misidentified it as E. coli
  • 16S rRNA sequencing
Not providedNorway[19]
Healthy male admitted acute sepsis of unknown origin
  • MALDI-TOF misidentified it as E. coli
  • 16S rRNA sequencing
Expanded-spectrum cephalosporinNorway[19]
66-year-old male with a history of gallstone disease developed acute postoperative sepsis with multiple organ failure.
  • MALDI-TOF misidentified it as E. coli
  • 16S rRNA sequencing
Intravenous piperacillin–tazobactam followed by oral trimethoprim–sulfamethoxazoleNorway[19]
Patient had Urinary tract infections
  • MALDI-TOF
  • 16S rRNA sequencing
Not providedAustralia[20]
64-year-old man undergoing chemotherapy for urinary tract cancer was hospitalized with febrile neutropenia.
  • MALDI-TOF misidentified it as E. coli
  • 16S rRNA sequencing
Patient diedCzech Republic[21]
Sepsis
  • MALDI-TOF misidentified it as E. coli
  • Confirmed with Whole-Genome Sequencing (WGS).
Not providedU.S.[22]
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Oladipo, P.; Kade, A.; Onohuean, H.; Ram, J.L. From Cryptic Clade to Emerging Pathogen: Exploring the Evolutionary Divergence and Clinical Relevance of Escherichia marmotae. Microorganisms 2026, 14, 869. https://doi.org/10.3390/microorganisms14040869

AMA Style

Oladipo P, Kade A, Onohuean H, Ram JL. From Cryptic Clade to Emerging Pathogen: Exploring the Evolutionary Divergence and Clinical Relevance of Escherichia marmotae. Microorganisms. 2026; 14(4):869. https://doi.org/10.3390/microorganisms14040869

Chicago/Turabian Style

Oladipo, Pelumi, Ayomikun Kade, Hope Onohuean, and Jeffrey L. Ram. 2026. "From Cryptic Clade to Emerging Pathogen: Exploring the Evolutionary Divergence and Clinical Relevance of Escherichia marmotae" Microorganisms 14, no. 4: 869. https://doi.org/10.3390/microorganisms14040869

APA Style

Oladipo, P., Kade, A., Onohuean, H., & Ram, J. L. (2026). From Cryptic Clade to Emerging Pathogen: Exploring the Evolutionary Divergence and Clinical Relevance of Escherichia marmotae. Microorganisms, 14(4), 869. https://doi.org/10.3390/microorganisms14040869

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