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Molecular and in silico analyses for detection of Shiga toxin-producing Escherichia coli (STEC) and highly pathogenic enterohemorrhagic Escherichia coli (EHEC) using genetic markers located on plasmid, O Island 57 and O Island 71

Abstract

Background

Due to the diversity of Shiga toxin-producing Escherichia coli (STEC) isolates, detecting highly pathogenic strains in foodstuffs is challenging. Currently, reference protocols for STEC rely on the molecular detection of eae and the stx1 and/or stx2 genes, followed by the detection of serogroup-specific wzx or wzy genes related to the top 7 serogroups. However, these screening methods do not distinguish between samples in which a STEC possessing both determinants are present and those containing two or more organisms, each containing one of these genes. This study aimed to evaluate ecf1, Z2098, Z2099, and nleA genes as single markers and their combinations (ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA) as genetic markers to detect potentially pathogenic STEC by the polymerase chain reaction (PCR) in 96 animal samples, as well as in 52 whole genome sequences of human samples via in silico PCR analyses.

Results

In animal isolates, Z2098 and Z2098/Z2099 showed a strong association with the detected top 7 isolates, with 100% and 69.2% of them testing positive, respectively. In human isolates, Z2099 was detected in 95% of the top 7 HUS isolates, while Z2098/Z2099 and ecf1/Z2099 were detected in 87.5% of the top 7 HUS isolates.

Conclusions

Overall, using a single gene marker, Z2098, Z2099, and ecf1 are sensitive targets for screening the top 7 STEC isolates, and the combination of Z2098/Z2099 offers a more targeted initial screening method to detect the top 7 STEC isolates. Detecting non-top 7 STEC in both animal and human samples proved challenging due to inconsistent characteristics associated with the genetic markers studied.

Peer Review reports

Background

Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin-producing E. coli (STEC), cause severe human illnesses such as hemorrhagic colitis and hemolytic uremic syndrome (HUS) [1]. The contamination of food products by EHEC remains a global concern, potentially leading to outbreaks of human disease [2,3,4]. STEC comprises more than 400 serotypes, many of which have been reported in foodstuffs and animals. However, in most cases, severe human illness is attributed to one of the seven well-defined EHEC serogroups, namely O26, O45, O103, O111, O121, O145, and O157 [5, 6]. These serogroups are considered adulterants in beef trim by the U.S. Department of Agriculture’s (USDA) Food Safety and Inspection Service (FSIS), which routinely conducts verification testing for these strains in domestic and imported beef manufacturing trimmings [7].

The top 7 EHEC serogroups typically possess the stx and eae genes and are classified as having high virulence potential [8, 9]. The ISO/TS 13,136 (EU) and MLG5B.05 (US) reference methods include an initial screening for the presence of the stx1/stx2 and eae genes. Presumptive-positive samples are then examined for genes associated with the top 5 (O157, O26, O103, O111, and O145) or top 7 serogroups, depending on whether the ISO/TS 13,136 or MLG5B.05 protocols are used, respectively [10, 11]. One of the main drawbacks of these reference methods is that many eae-negative STEC, enteropathogenic E. coli (EPEC), E. albertii, and free stx-converting phages can also react with these genetic targets, leading to false-positive results, especially in food, feces, and environmental samples [12,13,14]. Therefore, new genetic markers that specifically target EHEC (stx-positive and eae-positive strains of the top 7 E. coli serotypes) may improve the testing of food when this subpopulation of STEC must be detected in compliance with specific regulations, thereby reducing the rate of false positive results.

Various studies have reported potential genetic markers for EHEC. In 2014, Luedtke et al. at the U.S. Department of Agriculture evaluated the use of the EHEC-specific target E. coli attaching and effacing gene-positive conserved fragment 1 (ecf1) for the detection of EHEC directly from cattle feces [15]. The ecf1 gene is located on a unique conserved 5.6-kb fragment on the enterohemolysin-encoding plasmid of eae- and ehxA-positive STEC [16]. This fragment is part of the ecf operon, which consists of four genes (ecf1 to ecf4) and encodes four proteins involved in cell wall synthesis [17]. Other targets have been derived from several pathogenicity islands (PAIs) located on chromosomal regions. PAIs harbor genes that can serve as genetic signatures. The suitability of Z2098 and Z2099 genes, on the genomic O island 57 (OI-57), which may be associated with increased virulence of STEC strains in humans [18], has been tested for the identification of human-virulent STEC strains, particularly those of the top 7 EHEC serotypes [5]. Another chromosomally located gene, nleA (espI) on O island 71 (OI-71), has also been proposed as a candidate to distinguish EHEC from EPEC and STEC strains that may not be associated with severe and epidemic diseases [19].

The ecf1, Z2098, Z2099, and nleA genes may indeed have intrinsic value in identifying the top 7 EHEC [20]. The association of such genetic markers with highly pathogenic EHEC strains would be of interest to increase the specificity of the EHEC screening step in food, fecal, and environmental samples [21]. To clarify this relationship, we identified the presence of each of these genetic markers in STEC and EHEC strains belonging to different serogroups associated with animal hosts in Iran. Furthermore, we evaluated their combinations to find the best approach for a more specific and sensitive detection of the top 7 EHEC strains. Additionally, in silico PCR (polymerase chain reaction) analyses were conducted on whole-genome sequences of the top 7 and other emerging STEC/EHEC serotypes in human HUS patients retrieved from the NCBI GenBank database to explore the suitability of this approach for application to culture-independent NGS-based methods.

Methods

STEC isolates investigated

A total of 50 STEC collection strains were utilized. These isolates were gathered from various provinces in Iran, including Tehran, Razavi Khorasan, Semnan, Mazandaran, and Khuzestan. They were obtained from different animal hosts (cattle, sheep, goats, and pigeons) during the period from 2018 to 2020, as documented in our previous studies (refer to Table S1 in the supplementary materials). Moreover, 46 new STEC strains were added to this study. These strains were collected from a total number of 75 fecal samples from cattle, 70 from sheep, and 15 from goats in Razavi Khorasan province during the period from April 2022 to June 2022 (Strains’ features are provided in Table S1).

DNA extraction

All E. coli isolates were confirmed through culturing on MacConkey agar, Eosin-Methylene Blue (EMB) agar and subsequent biochemical tests. Afterward, a pure colony of each isolate was cultured on Luria Bertani (LB) agar and incubated for 24 h at 37 °C. After overnight culture on LB agar, total genomic DNA was extracted by the boiling method. In brief, a loopful from confluent growth area in LB agar culture was suspended in sterile microtubes containing 350 µL molecular grade water and boiled for 10 min. Then, samples were centrifuged at 5000×g for 5 min, and the supernatants were used as templates for end-point PCR assays. The quality and quantity of the extracted total genomes were evaluated using the NanoDrop-1000 spectrophotometer (ThermoFisher). DNA templates were kept at − 20 °C until the further analyses.

Strain characterization

To confirm the STEC genotype, a multiplex-PCR (Table 1) targeting stx1, stx2, eae, and ehxA was used as described previously [22]. For detection of stx in pigeon isolates, a single primers pair was used (Table 1) to amplify the stx2f [23]. E. coli O157:H7 strain (ATCC 35218) and E. coli O157:H7 strain Sakai (ATCC BAA-460) were used as positive controls. The strain PG90 from the Ferdowsi University of Mashhad (FUM) collection was included as a control for stx2f.

Table 1 Primers used for identification of virulence/genetic markers and serogroups of studied STEC/EHEC isolates

Based on the data collected annually by the European Centre for Disease Prevention and Control (ECDC), the 14 serogroups more frequently associated with the disease in humans in the EU include O26, O45, O55, O80, O91, O103, O104, O111, O113, O121, O128, O145, O146, and O157 [24]. Serogroups O26, O45, O55, O91, O103, O104, O111, O113, O121, O128, and O145 were determined [25] by PCR using the primers shown in Table 1 [26,27,28]. Additionally, the serogroups of O80 and O146 were tested as described elsewhere [29]. E. coli O157:H7 (295 EC-TMU) and FUM collection strains were used as positive controls.

PCR for ecf1 gene

A PCR assay (Table 1), was used to amplify the ecf1 gene as described previously [30]. Amplification was performed in a final volume of 20 µL containing 2 µL template DNA, 1 unit Taq DNA polymerase, 0.5 µM of each primer, 1.5 mM MgCl2, and 200 µM dNTP mix in 1× PCR buffer as follows: initial denaturation at 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 57 °C for 50 s and 72 °C for 30 s; and a final elongation step at 72 °C for 5 min. Amplicons were visualized after running at 100 V for 45 min on a 1.5% agarose gel containing green viewer safe stain. A 100 bp DNA ladder (CinnaGen, Iran) was used as a size marker. E. coli O157:H7 strain Sakai (ATCC BAA-460) was used as positive control in every PCR reaction (Table 1).

PCR for O Island 57 markers (Z2098 and Z2099 genes)

Amplification of Z2098 and Z2099 genes was carried out by a duplex-PCR assay (Table 1) according to our previous study [31]. Total DNA (2 µL) was used as template in a final volume of 25 µL mixture containing, 1 unit Taq DNA polymerase, 0.75 µM of each primer, 1.5 mM MgCl2, and 200 µM dNTP mix in 1× PCR buffer as follows: initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 40 s, 56 °C for 30 s and 72 °C for 45 s; and a final elongation step at 72 °C for 7 min. Amplicons were visualized after running at 100 V for 1 h on a 1.5% agarose gel containing green viewer safe stain. A 100 bp DNA ladder (CinnaGen, Iran) was used as a size marker. The positive control was E. coli O157:H7 strain Sakai (ATCC BAA-460) that was used in every PCR reaction.

PCR for O island 71 marker (nleA gene)

The presence of nleA gene was evaluated using the uniplex-PCR (Table 1) designed by Mundy et al. [32]. PCR was carried out in 20 µL using 2 µL template DNA, 1 unit Taq DNA polymerase, 0.5 µM of each primer, 1.5 mM MgCl2, and 200 µM dNTP mix in 1× PCR buffer as follows: initial denaturation at 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 52 °C for 55 s and 72 °C for 1 min; and a final elongation step at 72 °C for 5 min. Amplicons were visualized after running at 100 V for 45 min on a 1.5% agarose gel containing green viewer safe stain. A 100 bp DNA ladder (CinnaGen, Iran) was used as a size marker. E. coli O157:H7 strain Sakai (ATCC BAA-460) was used as positive control.

In silico PCR evaluation of genetic marker combinations in the top 7 and other important STEC/EHEC serotypes related to HUS patients

A total of 52 complete genome sequences of the top 7 (n = 40) and other emerging (n = 12) STEC/EHEC serotypes originating from human HUS patients were retrieved from NCBI GenBank database [33,34,35,36] (Accession numbers are provided in Table 2). Primer sequences of the genetic marker combinations, ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA were used to blast the genomes of the studied isolates to find the exact annealing sites and to extract the sequences corresponding the amplification products using CLC Genomics Workbench version 20.0 (https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-clc-genomics-workbench/). The complete genome sequence of strain Sakai (ATCC BAA-460) was used as a reference.

Table 2 Metadata of the 52 whole genomes of the top 7 (n = 40) and other important (n = 12) EHEC serotypes originating from HUS patients

Results

STEC/EHEC serogroups of animal strains

Overall, the 96 animal STEC/EHEC isolates belonged to the serogroups O5 (n = 13), O26 (n = 6), O80 (n = 2), O91 (n = 3), O103 (n = 12), O111 (n = 3), O113 (n = 13), and O128 (n = 9). For 35 isolates, the O-groups were not defined based on the included serogroups surveyed.

STEC/EHEC serotypes of human genomes

The 52 whole genome sequences retrieved from the GenBank belonged to serotypes O26:H11 (n = 4), O45:H2 (n = 2), O45:H16 (n = 1), O103:H2 (n = 1), O103:H8 (n = 1), O103:H11 (n = 1), O103:H25 (n = 1), O111:H8 (n = 2), O121:H19 (n = 10), O145:H25 (n = 2), O145:H28 (n = 3), O157:H7 (n = 12), O10:H25 (n = 1), O25:H4 (n = 1), O55:H7 (n = 2), O59:H19 (n = 1), O78:H4 (n = 1), O104:H4 (n = 1), O109:H21 (n = 1), O146:H28 (n = 1), O165:H25 (n = 2), and O182:H25 (n = 1).

Distribution of ecf1, Z2098, Z2099, and nleA in Animal STEC/EHEC strains

Overall, the genetic marker ecf1 was detected in eight isolates: four O26 (4/6, 66.6%), three O111 (3/3, 100%), and one isolate for which the serogroup could not be identified (Fig. 1). All the eight ecf1-positive isolates were identified as EHEC strains (stx and eae positive) (8 of 32 EHEC, 25.0%) (Fig. 2). The genetic marker Z2098 was present in 15 isolates: five strains of each of the O26 (5/6, 83.3%) and O103 (5/12, 41.6%) serogroups, three O111 (3/3, 100%), and two not defined serotypes (Fig. 1). Of the 15 Z2098-positive isolates, 9 strains were identified as EHEC (9/32, 28.1%) (Fig. 2). The Z2099 genetic marker was detected in 38 isolates belonging to serogroups O5 (12 strains, 12/13, 92.3%), O26 (5 strains, 5/6, 83.3%), O111 (three strains, 3/3, 100%), O91 (three strains, 3/3, 100%), two isolates of each O113 (2/13, 15.3%) and O103 (2/12, 16.6%), and in 11 strains of not defined serogroup (Fig. 1). Among the 38 Z2099-positive isolates, 9 strains were identified as EHEC (9/32, 28.1%) (Fig. 2). The nleA genetic marker was detected in six isolates: five strains of O26 (5/6, 83.3%), and one isolate of a not defined serogroup (Fig. 1). All the nleA-positive isolates were identified as EHEC (6/32, 18.7%) (Fig. 2). More details are provided in Table 3.

Table 3 Presence of genetic/virulence markers in 42/96 STEC and EHEC isolates included in this study
Fig. 1
figure 1

Distribution of the genetic markers ecf1, Z2098, Z2099, and nleA in different STEC/EHEC serogroups. Isolates lacking defined serogroups were excluded

Fig. 2
figure 2

Distribution of the genetic markers ecf1, Z2098, Z2099, and nleA in STEC and EHEC isolates

The genetic markers were also explored to identify combinations possibly associated with the top 7 EHEC ensuring a better sensitivity and specificity. As shown in Table 4, we detected the combined presence of Z2098/Z2099 with 100% frequency rate for O26, O111, O157 and 40.0% for O103; ecf1/Z2098 and ecf1/Z2099 presented a 100% distribution in the O111 and O157 strains and genomes tested, and 80% in O26; Z2098/nleA and Z2099/nleA combination showed 100% presence in O26 and O157; and ecf1/nleA a 100% distribution in O157 and 80% in O26. Interestingly, the non-top 7 isolates (O113, O5, and O91) were negative for all of these genetic marker combinations.

Table 4 Frequencies of the genetic marker combinations according to the top 7 and other important serogroups in studied STEC/EHEC isolates

In silico PCR analyses for distribution of the single genetic markers and their combinations in the top 7 and emerging STEC/EHEC serotypes in HUS patients

In total, the ecf1, Z2098, Z2099, and nleA were present as single genetic markers in 75.0%, 69.2%, 78.8%, and 53.8% of all the 52 STEC/EHEC serotypes related to HUS patients. Among these, Z2099 showed the highest presence (95%) in the top 7 EHEC serotypes and nleA had the lowest distribution (70%). For non-top 7 serotypes, ecf1 was detected as the most abundant single genetic marker in 33.3%, while none of the non-top 7 isolates were positive for nleA marker (Table 2).

Distribution of the genetic marker combinations ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA among the different genomes of STEC/EHEC serotypes is shown in Table 5. Overall, the combinations of Z2098/Z2099 (77.0%) and ecf1/Z2099 (77.0%) had the highest frequency rate among the top 7 EHEC serotypes while Z2098/nleA (31.2%) were observed as the lowest. Interestingly, the genetic markers investigated were less commonly associated with non-top 7 serotypes; we only detected ecf1/Z2099 in O165:H25 and ecf1/Z2098, ecf1/Z2099, and Z2098/Z2099 combinations in O182:H25 (Tables 5 and 2). The protocol chart in Fig. 3 illustrates the summary of methods and results.

Table 5 Distribution of the genetic marker combinations in the top 7 and other important STEC/EHEC serotypes related to HUS patients based on the in silico PCR analyses
Fig. 3
figure 3

The proposed single and combination of markers for searching the possible pathogenic strains in two categories of isolates (Animal STEC, human HUS) based on the findings of the current study

Discussion

Rapid and specific detection of EHEC strains is urgently needed by public health authorities to establish monitoring programs that track EHEC contamination in animals and foodstuffs. Generally, the ISO/TS 13,136 and MLG5B.05 reference methods rely on the presence of eae and the stx1 and/or stx2 genes, followed by the detection of O antigen genes (wzx or wzy) related to the top 5 and top 7 serogroups, respectively. These methods include screening for the detection of two genetic markers (eae and stx) in enrichment cultures, and presumptive positive results can be obtained from samples containing two or more organisms, each containing one of these genes [10, 11]. To this end, we examined the use of ecf1, Z2098, Z2099, and nleA genes as single markers, and ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA as genetic marker combinations to characterize a panel of STEC strains of animal origin and genomes from human cases of HUS to identify possible markers for the direct screening of food and animals for the presence of strains associated with severe human disease.

Z2098 and ecf1 as the best single genetic marker for screening of the top 7 EHEC/STEC isolates in animal hosts

In this study, we sought to investigate the use of genetic markers ecf1, Z2098, Z2099, and nleA to screen the top 7 and other STEC/EHEC serotypes originating from animal hosts. In this regard, we found a strong linkage between Z2098 and the top 7 STEC isolates, with an association of 100% (O26, O103, O111). Another gene marker, Z2099, showed significant potential to identify the top 7 isolates, as 76.9% of the O26, O103, and O111 serogroups were positive for the presence of this gene. Importantly, Z2099 was also an excellent genetic marker for some emerging STEC isolates, as we detected the marker in 100% of the O113 STEC strains. Similar data were reported by Delannoy et al., who described that the Z2098 and Z2099 gene markers had a detection range of 89.6–95.5% for the top six serogroups and a range of 67.6–96.8% for emerging STEC from other serogroups [5]. Another gene marker, ecf1, was detected in 53.8% of the top 7 animal EHEC isolates (O26 and O111), while all of the non-top 7 serogroups were negative for the gene marker. The use of ecf1 as a genetic marker to detect STEC isolates was examined by Livezey et al. (2015), who reported that 94.8% of the top 7 STEC strains were ecf1 positive [30]. Although ecf1 was more specific for the top 7 serotypes, the low incidence (53.8%) found in this work places this marker as the second choice for screening the top 7 STEC/EHEC serotypes after the Z2098 gene marker. Moreover, ecf1 is a plasmid gene marker that might be lost, leading to false negative results. Our animal samples were also screened for the presence of the nleA gene marker. Based on the results, this gene seems to have low potential for use as a marker, since only 38.4% of the top 7 serotypes (O26) were positive for nleA, and all of the non-top 7 serogroups were negative. However, a study conducted in the UK reported that 86% of the EHEC isolates from the patients with HUS and diarrhea were positive for the nleA gene marker [32]. These differences in the results obtained might be linked to the source of the samples studied, as the UK study was conducted on human samples instead of animal isolates. Hence, we also surveyed all the studied markers in highly pathogenic EHEC isolates originating from HUS patients via in silico PCR analyses to generate a comparison with animal isolates. Moreover, it has been reported that the nleA gene marker is associated with O26:H11, which confirms our results, as all of the nleA-positive isolates in our study were of the O26 serogroup [37, 38].

Z2098/Z2099 as the best genetic marker combination for detecting of the top 7 EHEC/STEC isolates in animal hosts

Our study showed that the combinations of the genetic markers ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA were detected in 53.8%, 53.8%, 30.7%, 69.2%, 38.4%, and 38.4% of the top 7 EHEC/STEC isolates, respectively. To date, this is the first report on the evaluation of these genetic marker combinations as a tracking method for the top 7 EHEC/STEC isolates of animal origin. None of the combinations identified all the top 7 isolates, but Z2098/Z2099 showed a percentage of 69.2% for the top 7 EHEC/STEC isolates. Nevertheless, Z2098/Z2099 and other studied combinations were not capable of detecting non-top 7 isolates, especially the important serogroup O113, highlighting the need for complementary studies to investigate other genetic marker combinations for important non-top 7 O-groups.

Diagnostic application of single and combination of studied genetic markers in HUS isolates

In addition to animal hosts, the studied genetic markers were also investigated via in silico PCR analyses in the genomes of top 7 and emerging EHEC/STEC serotypes related to HUS patients. A comparison of the animal and human results revealed that among the studied markers, Z2099 is more prevalent in the top 7 HUS isolates, with 95% of the strains testing positive, whereas ecf1 and Z2098 were detected in 87.5% and nleA in 70% of the top 7 serotypes. Such data are in accordance with a study by Delannoy et al., which showed a prevalence rate of 87% for Z2098 and 91% for Z2099 in EHEC and EHEC-like strains [5]. Combinations of the genetic markers also revealed that Z2098/Z2099 and ecf1/Z2099 are the most prevalent double markers for detecting the top 7 HUS isolates, with a positive rate of 87.5%. Considering these points, none of the single genetic markers were capable of detecting all EHEC isolates; thus, Z2098/Z2099 or ecf1/Z2099 offers a better choice for identifying the highly pathogenic EHEC with greater confidence. However, since we failed to detect the presence of these markers in 5 out of 40 strains, these combinations need to be investigated in a much wider panel of isolates to confirm their suitability as markers for highly pathogenic STEC. In contrast to the top 7 isolates, the other STEC/EHEC serotypes studied were not identified by the genetic marker combinations. Only one STEC strain (O182:H25) was positive for ecf1/Z2098, Z2098/Z2099, and ecf1/Z2099. Among the single markers, ecf1 and Z2099 had a very low frequency (33.3% and 25%, respectively) in the studied non-top 7 STEC/EHEC strains, with none of them positive for the nleA gene marker. Our study pointed out the need for additional markers to be tested in future research to find more sensitive and specific gene markers for non-top 7 HUS isolates.

We did not investigate these markers and their combinations in other pathogenic E. coli such as EPEC and other E. coli pathogroups. However, in Delannoy’s report, the distribution of the studied markers was significantly more prevalent in EHEC than in EPEC and apathogenic E. coli [7]. In the investigation conducted by Delannoy et al., it was demonstrated that 23.2% of the examined EPEC isolates exhibited positivity for the Z2098 marker. This phenomenon is attributed to the presence of stx-negative variants of EHEC, particularly those belonging to the top 7 EHEC serotypes, as outlined by the authors. It is noteworthy that, to avoid presumptive positive results, we propose analyzing the single markers in enrichment cultures; if positive, testing for combination markers can be applied to pure isolates. This approach may be particularly suitable in low- and middle-income countries where NGS facilities to characterize isolated STEC strains are not widely available.

Conclusions

Our study identified alternative genetic markers (Z2098, Z2099, and ecf1) that are effective for screening the top 7 STEC/EHEC strains, providing a specific method for detection without relying on traditional stx and eae markers. However, these markers should be used on pure cultures to avoid false positives. Detecting emerging non-top 7 EHEC strains remains challenging, highlighting the need for further research to find additional markers for these strains.

Data availability

We confirm that all data and findings of this study are available within the Article/Supplementary material.

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Acknowledgements

We appreciate our colleagues and technicians in Escherichia coli Research Lab at Ferdowsi University of Mashhad, who assisted us in this research project.

Funding

This project was supported by Ferdowsi University of Mashhad under the Grant No. FUM. 57299.

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Authors

Contributions

A.N. collected samples, carried out the analysis of samples, data analysis, and wrote the manuscript. M.A., Gh.H. and S.M. designed the study, supervised the project, revised the data analysis, and critically revised all parts of the manuscript. A.D. formal analysis, writing—review and editing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Mahdi Askari Badouei.

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Ethics approval and consent to participate

All procedures involving animals and their care in this study were approved (No. IR140257299) by Iran National Committee for Ethics in Biomedical Research. Moreover, a written or verbal informed consent was obtained from all participants for human experimentation and verbal informed consent was obtained from the owners of the companion animals. The research committee of Ferdowsi University of Mashhad reviewed and approved that all the study protocols were conducted in accordance with the related guidelines and regulations (No. FUM57299). The study was carried out in accordance with the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357).

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The authors declare no competing interests.

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Nemati, A., Askari Badouei, M., Hashemi Tabar, G. et al. Molecular and in silico analyses for detection of Shiga toxin-producing Escherichia coli (STEC) and highly pathogenic enterohemorrhagic Escherichia coli (EHEC) using genetic markers located on plasmid, O Island 57 and O Island 71. BMC Vet Res 20, 413 (2024). https://doi.org/10.1186/s12917-024-04251-0

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