1. Introduction
Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of human gastrointestinal diseases ranging from mild, watery to bloody diarrhea. The severe complications associated with toxin production and release can cause the life-threatening hemolytic uremic syndrome (HUS), leading to kidney failure and neurological episodes [1]. It has been reported that 6–25% of patients infected with STEC develop HUS, and the rate is higher in children [2,3,4]. More than 1000 different serotypes of STEC have been defined in humans, animals and the environment [5,6,7]. O157:H7 is the predominant serotype causing more severe disease. However, non-O157 serogroups such as O26, O45, O103, O111, O121, and O145 (referred to as the ‘top six’ non-O157 STEC) have been increasingly reported in outbreaks and sporadic cases in recent years in different countries like Europe and North America [8,9,10].
Stx1 and Stx2 are the main virulence factors of STEC. There are at least three stx1 subtypes (stx1a, stx1c, and stx1d) and twelve stx2 subtypes (stx2a-stx2l), associated with different clinical outcomes and disease severity [11,12,13,14,15]. stx2a, stx2c, and stx2d subtypes are frequently related to a higher risk of HUS, whereas other subtypes such as stx2e, stx2b, stx2f, and stx2g are related to less severe illnesses [11,16,17]. Besides Stx, intimin encoded by eae is considered as an important virulence factor in STEC. eae resides on the locus of enterocyte effacement (LEE) pathogenicity island and implicates attaching and effacing lesions in intestinal cells, attributing to determining the course of STEC infections [18]. Based on the difference of intimin C-terminal where cellular binding activity is highly variable, more than 30 intimin subtypes have been identified [19], and the most common subtypes are α, β, γ, ε, ζ, and η [20]. Different eae subtypes are associated with host specificity and tissue sensitivity. eae-β has been shown to predominate in non-O157 STEC strains from diarrheal patients, and eae-γ1 is associated with severe symptoms [21]. eae-ζ tends to be present in isolates from cattle [22,23]. eae subtypes are often associated with particular serotypes or pathotypes [24]. The serotypes O157:H7 and O145:H28 are linked to the eae-γ1, whereas O26:H11, O103:H2, and O111:H8 tend to carry eae-β1, eae-ε, and eae-θ, respectively [25]. stx2 and eae are commonly found in STEC strains linked with severe clinical outcomes [26].
Studies on the molecular characteristics of eae in clinical STEC strains are limited, especially in pediatric patients, and the relationship between eae subtypes and clinical symptoms remains to be addressed. In this study, we investigated the subtypes and polymorphisms of eae among clinical STEC strains from pediatric patients under 17 years old in Finland, and assessed the association of eae subtypes with clinical symptoms (HUS and non-HUS).
2. Results
2.1. Prevalence of eae in STEC Isolates
Among 240 STEC isolates, eae was present in 209 (87.1%) strains, including 49 (94.2%) strains from HUS patients, and 160 (85.1%) from non-HUS patients. All O157:H7 strains (n = 126) and 83 out of 114 (72.8%) non-O157 strains harbored eae (Table 1). eae is significantly associated with O157:H7 strains (p < 0.0001) (Table 1). No association was observed between the presence of eae and HUS status, age, or sex (Table 1).
2.2. Diversity of eae
In total, 209 complete eae sequences were extracted from all eae-positive STEC genomes, among which 23 unique sequences were identified, with the nucleotide identities ranging from 90.45% to 99.98%. The 23 sequences were assigned into five eae subtypes, namely ε1, γ1, β3, θ, and ζ3 (Figure 1). eae-γ1 was present in 152 (72.7%) strains, being the most predominant subtype, followed by β3 (33, 15.8%) and ε1 (16, 7.7%). eae sequence polymorphism was further illustrated by genotypes (GTs) within a subtype. γ1 subtype showed the highest diversity with eight GTs (GT1-GT8), followed by β3 (GT1-GT6), ε1 (GT1-GT4), θ (GT1-GT4), and ζ3 (GT1) (Figure 1). Using BLASTn search against the GenBank database (nr/nt), 13 eae genotypes showed 100% nucleotide identity to the publicly available eae sequences, while 10 eae genotypes (γ1/GT2, γ1/GT7, γ1/GT8, β3/GT3, β3/GT4, β3/GT5, β3/GT6, ε1/GT3, θ/GT2, and θ/GT4) showed 99.89–99.96% nucleotide identity to the publicly available sequences in the database (accessed 17/8/2023). Notably, γ1 was statistically significantly overrepresented in strains from patients aged 5 to 17 years (p < 0.0001), while β3 and ε1 were significantly higher in strains from patients under 5 years (p = 0.001 and p = 0.037) (Table 2). No statistically significant difference was found between different eae subtypes and HUS status.
2.3. Association of eae Subtypes/Genotypes with Serotypes
Nineteen serotypes were identified among 209 eae-positive STEC strains. O157:H7 was the most predominant serotype (126 strains, 60.3%), followed by O26:H11 (25, 12.0%), O145:H28 (17, 8.1%), O103:H2 (10, 4.8%), O55:H7 (8, 3.8%), O121:H19 (5, 2.4%), O111:H8 (4, 1.9%), and O5:H9 (3, 1.4%). All O157:H7 strains carried eae-γ1; among non-O157 strains, strains of each serotype harbored one eae subtype (Table S2). Strains of serotype O145:H28 and O55:H7 carried eae-γ1; O26:H11, O26:H21, O5:H9, O51:H49, O69:H11, O177:H25, and O151:H16 strains carried eae-β3; O103:H2, O121:H19, and O123:H2 strains carried eae-ε1; O111:H8 and O10:H25 strains carried eae-θ; and O156:H25, O182:H25, and O84:H2 strains carried eae-ζ3 (Table S2). eae-γ1 was statistically significantly overrepresented in O157:H7 strains (p < 0.0001), while eae-ε1, eae-β3, and eae-θ were more prevalent in non-O157 strains (p < 0.0001) (Table 2). Among the six clade 8 O157:H7 strains, two strains from patients with HUS harbored eae-γ1/GT6 (Figure 1).
2.4. Characterization of stx Subtypes in eae-Positive STEC Isolates in Relation to HUS
One stx1 subtype (stx1a) and three stx2 subtypes (stx2a, stx2c, and stx2e) were identified in 209 eae-positive STEC strains. Six stx subtypes combinations were present, including stx2a (108 strains), stx1a+stx2c (46), stx1a (39), stx1a+stx2a (9), stx2c (6), and stx2e (1). stx2 (115, 55.0%) was the most prevalent, especially in strains from HUS patients (46, 93.9%), and stx2a (108, 51.7%) was the most predominant stx subtype (Table 3 and Table S3). An association was observed between stx subtypes and eae subtypes. All strains carrying stx1a+stx2c harbored eae-γ1, and eae-γ1 was also overrepresented in strains carrying stx2a (p = 0.0049). eae-γ1 was less prevalent in strains with stx1a (p < 0.0001), while eae-β3 was less prevalent in strains with stx1a+stx2c (p = 0.0002). eae-β3, eae-ε1, eae-θ, and eae-ζ3 were more prevalent in strains with stx1a (p < 0.0001, p < 0.0001, p = 0.0046, and p = 0.0061, respectively) (Table 2 and Table S3).
An association between stx subtypes and HUS status or age was analyzed in the presence of eae. stx1a+stx2c+eae-γ1 and stx1a+eae-β3 were more prevalent in non-HUS-associated strains (p < 0.0001and p = 0.0245), while stx2a+eae-γ1 was significantly associated with HUS (p < 0.0001). In addition, stx1a+stx2c+eae-γ1 was more prevalent in strains from patients aged 5–17 than in those under 5 years (p = 0.0071) (Table 3).
Additionally, the relationship between a combination of stx+eae+serotypes (O157 and non-O157) and HUS status was evaluated. stx2a+eae-γ1+O157:H7 was strongly associated with HUS, while stx1a+eae-β3+non-O157 and stx1a+stx2c+eae-γ1+O157:H7 were associated with non-HUS (Table S4).
2.5. Comparison with eae-Positive STEC Strains from Swedish Pediatric Patients
In a previous study in Sweden [21], eae-γ1 was found to be statistically overrepresented in clinical eae-positive STEC strains from HUS patients, while β3 was significantly higher in non-HUS STEC strains from both adults and children. In addition, eae was found to be more prevalent in strains from children; however, the relationship between eae subtypes and clinical outcomes in strains from children was unclear in Sweden. We were interested to know if the correlation between eae subtype and clinical outcome was age-dependent. We then extracted 110 eae-positive strains from Swedish patients under 17 years old, and performed the same statistical analysis on the Swedish strains, as well as all 319 strains, including 110 from Swedish patients and 209 from Finnish patients within the same age group (under 17 years). The results showed that the presence of eae and the eae-γ1 subtype was significantly higher in strains from patients with HUS compared to strains from patients without HUS under 17 years old in Sweden (p = 0.0005 and p < 0.0001) (Table 4). The same correlation was observed in merged strains from Sweden and Finland under 17 years old (Table S5). eae-β3 was significantly higher in strains from non-HUS-STEC patients in Sweden, and in merged non-HUS-STEC strains from the two countries compared to HUS-STEC strains (p = 0.0001 and p = 0.0033) (Table 4 and Table S5).
3. Discussion
Over 90% of pediatric HUS cases are caused by gastrointestinal infection with STEC [2,27,28]. The reasons why children are more commonly affected than adults are unknown, but potential explanations may relate to the ability of specific strains to establish disease in specific populations, the age-specific expression on cells of receptors for toxins, or diameters of renal blood vessels. The presence of eae has been reported to be significantly associated with disease severity, and has previously been identified as a risk factor for the development of HUS. However, information about the characteristics and genetic diversity of eae and their contribution and correlation to disease severity is currently limited, especially in pediatric patients. In this study, eae was present in 87.1% of clinical pediatric strains, in accordance with previous studies, revealing that most clinical STEC strains possess eae [29,30]. The majority of eae-positive strains were of serotype O157:H7 (60.3%). It is worth noting that 72.8% of non-O157 strains in this study were eae positive, which was higher than previous findings reported in England (52.5%) [31] and Sweden (62.1%) [21]. However, the prevalence was reported in strains from children and adult patients together. This may indicate that eae is more prevalent in STEC strains from children than adults. The difference may be also due to the variations between geographical regions, sex, etc.
Various eae subtypes may confer distinct colonization patterns within the human intestine, thus leading to distinct pathogenic capability. STEC strains with eae-β, ε, γ1, θ, and ζ subtypes have been reported to be more virulent, and thus pose a higher pathogenic risk [21,25]. Previously, eae-γ1-positive STEC strains were isolated from children with HUS in Uruguay, highlighting the clinical significance of eae-γ1 [32]. In this study, five eae subtypes were identified (eae-γ1, β3, ε1, θ, and ζ3), of which eae-γ1 was the most prevalent, followed by β3 and ε1. In addition, eae-γ1 was more prevalent in children aged 5–17 years compared to children below 5 years, while β3 and ε1 were more prevalent in strains from children under 5 years, indicating that various eae subtypes tended to be present in different age groups. eae-γ1 was significantly associated with O157:H7 strains. Consistent with the previous findings, we found that eae-γ1 was carried by serogroup O157 and O145 strains; eae-β3 was carried by O26 strains; eae-ε1 was carried by O103 and O121 strains; and eae-θ was carried by O111 strains [25]. Certain eae subtypes may play roles in the pathogenicity of these clinical high-risk strains, leading to different clinical manifestations. No association was found between the presence of eae/its subtypes and HUS status in Finland. Interestingly, the presence of eae and eae-γ1 was significantly associated with HUS in patients within the same age in Sweden.
STEC strains producing Stx2a or Stx2c subtypes are more associated with HUS in humans, especially with the presence of eae [33]. We found that the presence of stx2a+eae-γ1 was strongly associated with HUS, while stx1a+eae-β3 and stx1a+stx2c+eae-γ1 were associated with non-HUS. This supports that the combination of stx2a+eae (particularly eae-γ1) can be used in risk assessments for the development of HUS and severe disease outcomes.
To conclude, our study showed high prevalence (87.1%) and genetic diversity of eae in clinical pediatric STEC strains. No correlation was observed between the presence of the eae gene/its subtypes and HUS status in pediatric STEC strains in Finland, while the combination of stx2a+eae-γ1 was significantly higher in STEC strains from patients with HUS.
4. Materials and Methods
4.1. Collection of eae-Positive STEC Isolates and Metadata
STEC strains were collected from STEC-infected pediatric patients under 17 years old in Finland between 2000 and 2016, and the clinical information was retrieved from the medical records as previously described [34]. Patients were classified into HUS and non-HUS groups according to clinical manifestation. Whole genome sequencing (WGS), assembly, and annotation were carried out as previously reported [35]. Genetic characterization of STEC strains, including stx subtyping, determination of serotypes, the presence of intimin encoding gene eae, and the clade-8 specific SNP were carried out as described previously [35]. Metadata of all STEC isolates, including strain ID, serotype, clade 8, presence of eae gene, eae subtype and genotype, stx subtype, and accession numbers of genomes, as well as clinical information of patients, including HUS status, age, and sex, are listed in Supplementary Table S1.
To examine the relationship between eae subtypes and HUS status in pediatric patients in different countries, STEC strains from STEC-infected patients under 17 years old in Sweden reported previously [21] were extracted and analyzed.
4.2. eae Subtyping
According to the genome annotation, complete eae sequences were extracted from the genome assemblies of 209 eae-positive STEC isolates, and then aligned with reference nucleotide sequences of eae subtypes downloaded from GeneBank. MEGA 11 software (Center for Evolutionary Medicine and Informatics, Tempe, AZ, USA) was used to compute the genetic distances of the eae subtypes with the maximum composite likelihood method, and a neighbor-joining phylogenetic tree was generated using 1000 bootstrap resampling. The phylogenetic tree structure and genetic distance were employed to determine the eae subtype. The diversity within each eae subtype was determined by eae GTs based on eae sequence polymorphism, as previously described [23].
4.3. Statistical Analyses
A statistical association between the existence of eae/its subtypes and strain characteristics (serotypes, stx subtypes) or clinical outcomes (HUS and non-HUS) was assessed with Fisher’s exact test using R software version 4.3.1 (
Conceptualization, A.M. and X.B.; methodology, L.W. and J.Z.; software, L.W.; investigation, A.M., L.W. and X.B.; resources, A.M., E.Y. and H.S.; writing—original draft preparation, L.W. and X.B.; writing—review and editing, L.W., X.B., E.Y., J.Z., H.S. and A.M.; supervision, X.B. and A.M.; project administration, A.M. and X.B.; funding acquisition, A.M. and X.B. All authors have read and agreed to the published version of the manuscript.
The use of patients’ information and the study protocol were approved by the Ethics Committee of the University of Helsinki (HUS/1274/2017) and date of approval was 20 April 2017.
Patient consent was waived due to retrospective review of the patients’ medical records. Patient data were anonymous, and no consent was required to work with the bacterial strains.
The assemblies of all strains in this study were deposited in GenBank with accession numbers and metadata shown in
We thank Saara Salmenlinna and Jani Halkilahti from the Department of Health Security, Finnish Institute for Health and Welfare, Helsinki, Finland, for support with bacterial strains and intellectual input on the work.
The authors declare no conflict of interest.
Footnotes
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Enlarge this image.Figure 1. Phylogenetic relationships of 23 unique eae sequences identified in this study (indicated in bold and different colors) and 30 reference sequences representing different eae subtypes based on the neighbor-joining method. For the branches that represent the identified 23 eae subtypes in this study, the tips are labeled in the order of eae subtype/genotype, serotype, stx subtype, and HUS status. The number of the strains are shown in brackets. Scale bar indicates genetic distance.
Prevalence of eae in 240 STEC strains isolated from pediatric patients (<17 years) #.
| eae | Serotype | p-Value | Clinical Symptom | p-Value | Age Group | p-Value | Sex | p-Value | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| O157:H7 |
Non-O157 |
HUS |
Non-HUS |
<5 Years |
5–17 Years |
Male |
Female |
|||||
| Positive | 126 (100.0) | 83 (72.81) | <0.0001 * | 49 (94.23) | 160 (85.13) | 0.1025 | 102 (87.18) | 107 (86.99) | 1 | 103 (84.43) | 106 (89.83) | 0.2502 |
# The association was analyzed between eae gene and serotypes (O157 and non-O157), clinical symptoms (HUS and non-HUS), age groups (<5 years and 5–17 years; the age was grouped according to the median age of 5 years), sex (male and female). The number represents the number of eae-positive strains, and percentage was shown in brackets. * Statistically significant difference.
Association between eae subtypes and serotypes or age or stx subtypes #.
| eae Subtype (No. of Strains) | Serotype | Age Group | stx Subtype | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| O157:H7 (n = 126) | Non-O157 (n = 83) | p-Value | <5 Years |
5–17 Years |
p-Value | stx2a |
p-Value | stx1a |
p-Value | stx1a+stx2c |
p-Value | ||||||||
| Pos | Prevalence | Pos | Prevalence | Pos | Prevalence | Pos | Prevalence | Pos | Prevalence | Pos | Prevalence | Pos | Prevalence | ||||||
| γ1 (152) | 126 | 100.0% | 26 | 31.33% | <0.0001 * | 60 | 58.82% | 92 | 85.98% | <0.0001 * | 88 | 81.48% | 0.0049 * | 7 | 17.95% | <0.0001 * | 46 | 100.00% | <0.0001 * |
| β3 (33) | 0 | 0.00% | 33 | 39.76% | <0.0001 * | 25 | 24.51% | 8 | 7.48% | 0.001 * | 14 | 12.96% | 0.261 | 15 | 38.46% | <0.0001 * | 0 | 0.00% | 0.0002 * |
| ε1 (16) | 0 | 0.00% | 16 | 19.28% | <0.0001 * | 12 | 11.76% | 4 | 3.74% | 0.037 * | 5 | 4.63% | 0.1187 | 10 | 25.64% | <0.0001 * | 0 | 0.00% | 0.0255 |
| θ (5) | 0 | 0.00% | 5 | 6.02% | 0.0092 * | 3 | 2.94% | 2 | 1.87% | 0.6772 | 1 | 0.93% | 0.200 | 4 | 10.26% | 0.0046 * | 0 | 0.00% | 0.5883 |
| ζ3 (3) | 0 | 0.00% | 3 | 3.61% | 0.0613 | 2 | 1.96% | 1 | 0.93% | 0.6143 | 0 | 0.00% | 0.1111 | 3 | 7.69% | 0.0061 * | 0 | 0.00% | 1 |
# The association was analyzed between eae subtype and clinical symptoms (HUS and non-HUS), age groups (<5 years and 5–17 years; the age was grouped according to the median age of 5 years), serotypes (O157 and non-O157), sex (male and female), as well as stx subtypes (stx2a, stx1a, stx2c, stx2e, stx1a+stx2a, and stx1a+stx2c), only variables with statistically significant differences (p < 0.05) were shown in this table. * Statistically significant difference. pos: number of positive strains.
Association between stx+eae and HUS status or age or serotypes #.
| stx+eae | No. of Strains | Clinical Symptom | p-Value | Age Group | p-Value | Serotype | p-Value | |||
|---|---|---|---|---|---|---|---|---|---|---|
| HUS (n = 49) | Non-HUS (n = 160) | <5 Years (n = 102) | 5–17 Years (n = 107) | O157:H7 (n = 126) | Non-O157 (n = 83) | |||||
| stx1 + eae | 39 | 1 (2.04) | 38 (23.75) | 0.0002 * | 24 (23.53) | 15 (14.02) | 0.1092 | 0 (0.00) | 39 (46.99) | <0.0001 * |
| stx1a+eae-β3 | 15 | 0 (0.00) | 15 (9.38) | 0.0245 * | - | - | - | 0 (0.00) | 15 (18.07) | <0.0001 * |
| stx1a+eae-ε1 | 10 | 0 (0.00) | 10 (6.25) | 0.1212 | - | - | - | 0 (0.00) | 10 (12.05) | <0.0001 * |
| stx1a+eae-γ1 | 7 | 0 (0.00) | 7 (4.37) | 0.2033 | - | - | - | 0 (0.00) | 7 (8.43) | 0.0013 * |
| stx1a+eae-θ | 4 | 0 (0.00) | 4 (2.50) | 0.5750 | - | - | - | 0 (0.00) | 4 (4.82) | 0.0267 * |
| stx1a+eae-ζ3 | 3 | 1 (2.04) | 2 (1.25) | 0.5739 | - | - | - | 0 (0.00) | 3 (3.61) | 0.0667 |
| stx2 + eae | 115 | 46 (93.88) | 69 (43.13) | <0.0001 * | 61 (59.80) | 54 (50.47) | 0.2108 | 74 (58.73) | 41 (49.40) | 0.2028 |
| stx2e+eae-β3 | 1 | 0 (0.00) | 1 (0.63) | 1 | 1 (0.98) | 0 (0.00) | 0.4880 | 0 (0.00) | 1 (1.20) | 0.3971 |
| stx2c+eae-γ1 | 6 | 2 (4.08) | 4 (2.50) | 0.6267 | 2 (1.96) | 4 (3.74) | 0.6835 | 5 (3.97) | 1 (1.20) | 0.4059 |
| stx2a+eae-γ1 | 88 | 35 (71.43) | 53 (33.13) | <0.0001 * | - | - | - | - | - | - |
| stx2a+eae-β3 | 14 | 6 (12.24) | 8 (5.00) | 0.0997 | - | - | - | - | - | - |
| stx2a+eae-ε1 | 5 | 2 (4.08) | 3 (1.89) | 0.3341 | - | - | - | - | - | - |
| stx2a+eae-θ | 1 | 0 (0.00) | 1 (0.63) | 1 | - | - | - | - | - | - |
| stx1 + stx2 + eae | 55 | 2 (4.08) | 53 (33.13) | <0.0001 * | 17 (16.67) | 38 (35.51) | 0.0027 * | 52 (41.27) | 3 (3.61) | <0.0001 * |
| stx1a+stx2a |
9 | 2 (4.08) | 7 (4.38) | 1 | 3 (2.94) | 6 (5.61) | 0.4993 | 6 (4.76) | 3 (3.61) | 1 |
| stx1a+stx2c |
46 | 0 (0.00) | 46 (28.75) | <0.0001 * | 14 (13.73) | 32 (29.91) | 0.0071 * | 46 (36.51) | 0 (0.00) | <0.0001 * |
# The association was analyzed between eae+stx subtype and clinical symptoms (HUS and non-HUS), age groups, and serotypes. The number represents the number of strains carrying specific stx+eae subtypes, and percentage is shown in brackes. * Statistically significant difference. pos: number of positive strains.
Association between eae subtypes and HUS status in pediatric patients under 17 years old in Sweden #.
| eae Subtype | No. of Strains | HUS (34) | Non-HUS (76) | p-Value | ||
|---|---|---|---|---|---|---|
| Pos | Prevalence | Pos | Prevalence | |||
| γ1 | 40 | 22 | 64.71% | 18 | 23.68% | <0.0001 * |
| ε1 | 35 | 9 | 26.47% | 26 | 34.21% | 0.5091 |
| β3 | 28 | 1 | 2.94% | 27 | 35.53% | 0.0001 * |
| θ | 5 | 2 | 5.88% | 3 | 3.95% | 0.6436 |
| ζ3 | 1 | 0 | 0.00% | 1 | 1.32% | 1 |
| ρ | 1 | 0 | 0.00% | 1 | 1.32% | 1 |
| In total | 110 | 34 | 97.14% | 76 | 70.37% | 0.0005 * |
# The association was analyzed between eae/its subtype and clinical symptoms (HUS and non-HUS) in strains from pediatric patients in Sweden. * Statistically significant difference. pos: number of positive strains.
Supplementary Materials
The following supporting information can be downloaded at:
References
1. Majowicz, S.E.; Scallan, E.; Jones-Bitton, A.; Sargeant, J.M.; Stapleton, J.; Angulo, F.J.; Yeung, D.H.; Kirk, M.D. Global Incidence of Human Shiga Toxin-Producing Escherichia coli Infections and Deaths: A Systematic Review and Knowledge Synthesis. Foodborne Pathog. Dis.; 2014; 11, pp. 447-455. [DOI: https://dx.doi.org/10.1089/fpd.2013.1704] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24750096]
2. Joseph, A.; Cointe, A.; Mariani Kurkdjian, P.; Rafat, C.; Hertig, A. Shiga Toxin-Associated Hemolytic Uremic Syndrome: A Narrative Review. Toxins; 2020; 12, 67. [DOI: https://dx.doi.org/10.3390/toxins12020067] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31973203]
3. Smith, J.L.; Fratamico, P.M.; Gunther, N.W. Shiga Toxin-Producing Escherichia coli. Advances in Applied Microbiology; Elsevier: Amsterdam, The Netherlands, 2014; Volume 86, pp. 145-197. ISBN 978-0-12-800262-9
4. Bryan, A.; Youngster, I.; McAdam, A.J. Shiga Toxin Producing Escherichia coli. Clin. Lab. Med.; 2015; 35, pp. 247-272. [DOI: https://dx.doi.org/10.1016/j.cll.2015.02.004]
5. Mussio, P.; Martínez, I.; Luzardo, S.; Navarro, A.; Leotta, G.; Varela, G. Phenotypic and Genotypic Characterization of Shiga Toxin-Producing Escherichia coli Strains Recovered from Bovine Carcasses in Uruguay. Front. Microbiol.; 2023; 14, 1130170. [DOI: https://dx.doi.org/10.3389/fmicb.2023.1130170] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36950166]
6. Bettelheim, K.A.; Goldwater, P.N. A Compendium of Shigatoxigenic Escherichia coli: Origins, Bacteriological and Clinical Data on the Serogroups, Serotypes and Untypeable Strains of E. Coli Reported between 1980 and 2017, Excluding O157:H7. Adv. Microbiol.; 2019; 9, pp. 905-909. [DOI: https://dx.doi.org/10.4236/aim.2019.911056]
7. Malahlela, M.; Cenci-Goga, B.; Marufu, M.; Theirry, F.; Grispoldi, L.; Etter, E.; Kalake, A.; Karama, M. Occurrence, Serotypes and Virulence Characteristics of Shiga-Toxin-Producing Escherichia coli Isolates from Goats on Communal Rangeland in South Africa. Toxins; 2022; 14, 353. [DOI: https://dx.doi.org/10.3390/toxins14050353]
8. Kinnula, S.; Hemminki, K.; Kotilainen, H.; Ruotsalainen, E.; Tarkka, E.; Salmenlinna, S.; Hallanvuo, S.; Leinonen, E.; Jukka, O.; Rimhanen-Finne, R. Outbreak of Multiple Strains of Non-O157 Shiga Toxin-Producing and Enteropathogenic Escherichia coli Associated with Rocket Salad, Finland, Autumn 2016. Eurosurveillance; 2018; 23, 1700666. [DOI: https://dx.doi.org/10.2807/1560-7917.ES.2018.23.35.1700666] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30180926]
9. Valilis, E.; Ramsey, A.; Sidiq, S.; DuPont, H.L. Non-O157 Shiga Toxin-Producing Escherichia coli—A Poorly Appreciated Enteric Pathogen: Systematic Review. Int. J. Infect. Dis.; 2018; 76, pp. 82-87. [DOI: https://dx.doi.org/10.1016/j.ijid.2018.09.002]
10. Tseng, M.; Sha, Q.; Rudrik, J.T.; Collins, J.; Henderson, T.; Funk, J.A.; Manning, S.D. Increasing Incidence of Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) in Michigan and Association with Clinical Illness. Epidemiol. Infect.; 2016; 144, pp. 1394-1405. [DOI: https://dx.doi.org/10.1017/S0950268815002836]
11. Byrne, L.; Adams, N.; Jenkins, C. Association between Shiga Toxin–Producing Escherichia coli O157:H7 Stx Gene Subtype and Disease Severity, England, 2009–2019. Emerg. Infect. Dis.; 2020; 26, pp. 2394-2400. [DOI: https://dx.doi.org/10.3201/eid2610.200319]
12. Rauw, K.D.; Buyl, R.; Jacquinet, S.; Piérard, D. Risk Determinants for the Development of Typical Hemolytic Uremic Syndrome in Belgium and Proposition of a New Virulence Typing Algorithm for Shiga Toxin-Producing Escherichia coli. Epidemiol. Infect.; 2019; 147, e6. [DOI: https://dx.doi.org/10.1017/S0950268818002546] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30182864]
13. Friedrich, A.W.; Borell, J.; Bielaszewska, M.; Fruth, A.; Tschäpe, H.; Karch, H. Shiga Toxin 1c-Producing Escherichia coli Strains: Phenotypic and Genetic Characterization and Association with Human Disease. J. Clin. Microbiol.; 2003; 41, pp. 2448-2453. [DOI: https://dx.doi.org/10.1128/JCM.41.6.2448-2453.2003] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12791863]
14. Torti, J.F.; Cuervo, P.; Nardello, A.; Pizarro, M. Epidemiology and Characterization of Shiga Toxin-Producing Escherichia coli of Hemolytic Uremic Syndrome in Argentina. Cureus; 2021; 13, e17213. [DOI: https://dx.doi.org/10.7759/cureus.17213] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34540440]
15. Gill, A.; Dussault, F.; McMahon, T.; Petronella, N.; Wang, X.; Cebelinski, E.; Scheutz, F.; Weedmark, K.; Blais, B.; Carrillo, C. Characterization of Atypical Shiga Toxin Gene Sequences and Description of Stx2j, a New Subtype. J. Clin. Microbiol.; 2022; 60, e02229-21. [DOI: https://dx.doi.org/10.1128/jcm.02229-21]
16. Beutin, L.; Krüger, U.; Krause, G.; Miko, A.; Martin, A.; Strauch, E. Evaluation of Major Types of Shiga Toxin 2e-Producing Escherichia coli Bacteria Present in Food, Pigs, and the Environment as Potential Pathogens for Humans. Appl. Environ. Microbiol.; 2008; 74, pp. 4806-4816. [DOI: https://dx.doi.org/10.1128/AEM.00623-08] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18515483]
17. Friesema, I.; Van Der Zwaluw, K.; Schuurman, T.; Kooistra-Smid, M.; Franz, E.; Van Duynhoven, Y.; Van Pelt, W. Emergence of Escherichia coli Encoding Shiga Toxin 2f in Human Shiga Toxin-Producing E. Coli (STEC) Infections in the Netherlands, January 2008 to December 2011. Eurosurveillance; 2014; 19, 20787. [DOI: https://dx.doi.org/10.2807/1560-7917.ES2014.19.17.20787] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24821123]
18. Stevens, M.P.; Frankel, G.M. The Locus of Enterocyte Effacement and Associated Virulence Factors of Enterohemorrhagic Escherichia coli. Microbiol. Spectr.; 2014; 2, pp. 97-130. [DOI: https://dx.doi.org/10.1128/microbiolspec.EHEC-0007-2013] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26104209]
19. Ooka, T.; Seto, K.; Kawano, K.; Kobayashi, H.; Etoh, Y.; Ichihara, S.; Kaneko, A.; Isobe, J.; Yamaguchi, K.; Horikawa, K. et al. Clinical Significance of Escherichia albertii. Emerg. Infect. Dis.; 2012; 18, pp. 488-492. [DOI: https://dx.doi.org/10.3201/eid1803.111401]
20. Zhang, W.L.; Köhler, B.; Oswald, E.; Beutin, L.; Karch, H.; Morabito, S.; Caprioli, A.; Suerbaum, S.; Schmidt, H. Genetic Diversity of Intimin Genes of Attaching and Effacing Escherichia coli Strains. J. Clin. Microbiol.; 2002; 40, pp. 4486-4492. [DOI: https://dx.doi.org/10.1128/JCM.40.12.4486-4492.2002]
21. Hua, Y.; Bai, X.; Zhang, J.; Jernberg, C.; Chromek, M.; Hansson, S.; Frykman, A.; Yang, X.; Xiong, Y.; Wan, C. et al. Molecular Characteristics of Eae-Positive Clinical Shiga Toxin-Producing Escherichia coli in Sweden. Emerg. Microbes Infect.; 2020; 9, pp. 2562-2570. [DOI: https://dx.doi.org/10.1080/22221751.2020.1850182]
22. Xu, Y.; Bai, X.; Zhao, A.; Zhang, W.; Ba, P.; Liu, K.; Jin, Y.; Wang, H.; Guo, Q.; Sun, H. et al. Genetic Diversity of Intimin Gene of Atypical Enteropathogenic Escherichia coli Isolated from Human, Animals and Raw Meats in China. PLoS ONE; 2016; 11, e0152571. [DOI: https://dx.doi.org/10.1371/journal.pone.0152571] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27031337]
23. Yang, X.; Sun, H.; Fan, R.; Fu, S.; Zhang, J.; Matussek, A.; Xiong, Y.; Bai, X. Genetic Diversity of the Intimin Gene (Eae) in Non-O157 Shiga Toxin-Producing Escherichia coli Strains in China. Sci. Rep.; 2020; 10, 3275. [DOI: https://dx.doi.org/10.1038/s41598-020-60225-w] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32094410]
24. Creuzburg, K.; Middendorf, B.; Mellmann, A.; Martaler, T.; Holz, C.; Fruth, A.; Karch, H.; Schmidt, H. Evolutionary Analysis and Distribution of Type III Effector Genes in Pathogenic Escherichia coli from Human, Animal and Food Sources. Environ. Microbiol.; 2011; 13, pp. 439-452. [DOI: https://dx.doi.org/10.1111/j.1462-2920.2010.02349.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20880329]
25. Tostes, R.; Goji, N.; Amoako, K.; Chui, L.; Kastelic, J.; DeVinney, R.; Stanford, K.; Reuter, T. Subtyping Escherichia coli Virulence Genes Isolated from Feces of Beef Cattle and Clinical Cases in Alberta. Foodborne Pathog. Dis.; 2017; 14, pp. 35-42. [DOI: https://dx.doi.org/10.1089/fpd.2016.2199] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27854514]
26. Werber, D.; Fruth, A.; Buchholz, U.; Prager, R.; Kramer, M.H.; Ammon, A.; Tschäpe, H. Strong Association Between Shiga Toxin-Producing Escherichia coli O157 and Virulence Genes Stx2 and Eae as Possible Explanation for Predominance of Serogroup O157 in Patients with Haemolytic Uraemic Syndrome. Eur. J. Clin. Microbiol. Infect. Dis.; 2003; 22, pp. 726-730. [DOI: https://dx.doi.org/10.1007/s10096-003-1025-0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/14614596]
27. Luna, M.; Kamariski, M.; Principi, I.; Bocanegra, V.; Vallés, P.G. Severely Ill Pediatric Patients with Shiga Toxin-Associated Hemolytic Uremic Syndrome (STEC-HUS) Who Suffered from Multiple Organ Involvement in the Early Stage. Pediatr. Nephrol.; 2021; 36, pp. 1499-1509. [DOI: https://dx.doi.org/10.1007/s00467-020-04829-4]
28. Liu, Y.; Thaker, H.; Wang, C.; Xu, Z.; Dong, M. Diagnosis and Treatment for Shiga Toxin-Producing Escherichia coli Associated Hemolytic Uremic Syndrome. Toxins; 2023; 15, 10. [DOI: https://dx.doi.org/10.3390/toxins15010010]
29. Fierz, L.; Cernela, N.; Hauser, E.; Nüesch-Inderbinen, M.; Stephan, R. Characteristics of Shigatoxin-Producing Escherichia coli Strains Isolated during 2010–2014 from Human Infections in Switzerland. Front. Microbiol.; 2017; 8, 1471. [DOI: https://dx.doi.org/10.3389/fmicb.2017.01471]
30. Hoang Minh, S.; Kimura, E.; Hoang Minh, D.; Honjoh, K.; Miyamoto, T. Virulence Characteristics of Shiga Toxin-Producing Escherichia coli from Raw Meats and Clinical Samples. Microbiol. Immunol.; 2015; 59, pp. 114-122. [DOI: https://dx.doi.org/10.1111/1348-0421.12235]
31. Byrne, L.; Vanstone, G.L.; Perry, N.T.; Launders, N.; Adak, G.K.; Godbole, G.; Grant, K.A.; Smith, R.; Jenkins, C. Epidemiology and Microbiology of Shiga Toxin-Producing Escherichia coli Other than Serogroup O157 in England, 2009–2013. J. Med. Microbiol.; 2014; 63, pp. 1181-1188. [DOI: https://dx.doi.org/10.1099/jmm.0.075895-0]
32. Pérez, L.; Apezteguía, L.; Piñeyrúa, C.; Dabezies, A.; Bianco, M.N.; Schelotto, F.; Varela, G. Hemolytic Uremic Syndrome with Mild Renal Involvement Due to Shiga Toxin-Producing Escherichia coli (STEC) O145 Strain. Rev. Argent. Microbiol.; 2014; 46, pp. 103-106. [DOI: https://dx.doi.org/10.1016/S0325-7541(14)70056-2] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25011592]
33. Taghadosi, R.; Shakibaie, P.M.R.; Alizade, H.; Hosseini-nave, H.; Askari, A.; Ghanbarpour, R. Serogroups, Subtypes and Virulence Factors of Shiga Toxin-Producing Escherichia coli Isolated from Human, Calves and Goats in Kerman, Iran. Gastroenterol. Hepatol. Bed Bench; 2018; 11, 60. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29564067]
34. Ylinen, E.; Salmenlinna, S.; Halkilahti, J.; Jahnukainen, T.; Korhonen, L.; Virkkala, T.; Rimhanen-Finne, R.; Nuutinen, M.; Kataja, J.; Arikoski, P. et al. Hemolytic Uremic Syndrome Caused by Shiga Toxin–Producing Escherichia coli in Children: Incidence, Risk Factors, and Clinical Outcome. Pediatr. Nephrol.; 2020; 35, pp. 1749-1759. [DOI: https://dx.doi.org/10.1007/s00467-020-04560-0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32323005]
35. Bai, X.; Ylinen, E.; Zhang, J.; Salmenlinna, S.; Halkilahti, J.; Saxen, H.; Narayanan, A.; Jahnukainen, T.; Matussek, A. Comparative Genomics of Shiga Toxin-Producing Escherichia coli Strains Isolated from Pediatric Patients with and without Hemolytic Uremic Syndrome from 2000 to 2016 in Finland. Microbiol. Spectr.; 2022; 10, e0066022. [DOI: https://dx.doi.org/10.1128/spectrum.00660-22]
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.