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. 2026 Apr 2;16:15882. doi: 10.1038/s41598-026-45559-1

Integrated genomic and phenotypic characterization of antimicrobial resistance in Shiga toxin-producing Escherichia coli from asymptomatic carriers in Japan

Yumi Imai 1, Miki Okuno 1, Shunsuke Iriguchi 1, Hiroshi Kaneko 2, Toshio Sato 2, Akio Noguchi 2, Takeshi Yamamoto 1, Yoshitoshi Ogura 1,✉
PMCID: PMC13194739  PMID: 41927856

Abstract

Shiga toxin-producing Escherichia coli (STEC) carried by asymptomatic individuals constitute an important reservoir for community transmission, but large-scale analyses of antimicrobial resistance (AMR) in carrier-derived STEC remain limited. In this study, to obtain fundamental information relevant to the eradication of STEC carriage, we analyzed 495 STEC strains isolated from stool-screened asymptomatic carriers in Japan. All the isolates had been previously subjected to whole-genome sequencing and were classified into more than 100 serotypes, predominantly O156:H25 (13.5%), O174:H21 (7.7%), and O105:H7 (6.1%). Genome-based analysis revealed at least one antimicrobial resistance gene (ARG) in 83 isolates (16.8%), with aph(3″)-Ib, aph(6)-Id, sul2, tet(A), and blaTEM-1 being the most prevalent. Most carrier-derived STEC belonged to phylogroup B1, whereas ARGs were identified in isolates from diverse phylogenetic lineages. Comparative analysis revealed that the AMR profiles of carrier-derived STEC were similar to those of human commensal E. coli, whereas cattle commensal isolates exhibited a higher ARG prevalence. Phenotypic susceptibility testing revealed that resistance to ampicillin (n = 33) and tetracycline (n = 50) was the most common, whereas resistance to fluoroquinolones (n = 1) was infrequent and no resistance was observed to fosfomycin (n = 0) or amikacin (n = 0). Together, these findings provide a comprehensive overview of AMR in STEC from asymptomatic carriers in Japan and support the continued effectiveness of several antimicrobial agents while underscoring the importance of ongoing genomic surveillance.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-45559-1.

Keywords: Shiga toxin-producing Escherichia coli, Antimicrobial resistance, Genome, Asymptomatic carrier

Subject terms: Genetics, Microbiology, Molecular biology

Introduction

Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen that causes diarrhea and hemorrhagic colitis and can lead to severe complications such as hemolytic uremic syndrome (HUS)1. STEC occasionally causes outbreaks of varying sizes and is a particular public health concern in both developed and developing countries2. Infants and elderly individuals are especially susceptible to severe disease. STEC produces two types of Shiga toxin, Stx1 and Stx2, which are further classified into at least three (Stx1a, -c, and -d) and fifteen (Stx2a–o) subtypes, respectively3–7. Among these factors, the production of Stx2a is recognized as a major risk factor for severe clinical outcomes8,9. In addition, strains carrying the locus of enterocyte effacement (LEE), which encodes a type III secretion system, are more frequently associated with severe disease8. Most STEC strains belonging to O157 and the so-called “big 6” non-O157 serogroups—O26, O111, O103, O121, O145, and O45—are LEE positive10. In Europe, STEC producing Stx2d have also been reported to confer a high risk of HUS following Stx2a-producing strains, and this association has been described even among LEE-negative lineages11.

The use of antimicrobials for STEC infections remains controversial because certain antibiotics can induce toxin release and worsen clinical symptoms12,13. In Japan, fosfomycin and fluoroquinolones are typically selected for the treatment of STEC infections in both pediatric and adult patients14–18. However, the use of antimicrobials in the treatment of STEC infections is still debated, and no consensus has been reached in Japan17,19.

Ruminants such as cattle serve as major reservoirs of STEC, and infection occurs through consumption of undercooked meat or contaminated produce and water20. Owing to its high infectivity, STEC can also spread via secondary human-to-human transmission, leading to household outbreaks or institutional outbreaks in settings such as nursing homes and kindergartens21,22. To prevent secondary transmission, many countries impose restrictions on the activities of STEC-infected individuals until shedding becomes undetectable21. In Japan, employment restrictions are applied regardless of symptoms, particularly for food handlers and workers in welfare facilities, who are also required to undergo periodic stool screening several times a year23,24. STEC carriers are subjected to eradication treatments using antimicrobial or probiotic preparations. However, some individuals remain positive for extended periods, resulting in psychological distress, financial burden, and operational difficulties for workplaces25.

In our previous investigation of routine stool screening among food handlers and welfare facility employees in Japan, we reported an STEC positivity rate of 0.035%26. Among the carrier-derived isolates, only 7.1% were classified as high-risk STEC harboring both stx2a and LEE, whereas low-risk STEC lacking both factors accounted for 57.0%, and stx2d-positive strains were also infrequent (0.8%), highlighting the need for risk-based public health responses. Nevertheless, individuals identified as STEC carriers are legally prohibited from working as food handlers or in social welfare facilities and therefore undergo eradication therapy regardless of the pathogenicity of the strain. Although oral azithromycin has been reported to be effective for long-term carriers25, clear guidelines on the use of antimicrobial agents for the eradication of STEC carriage in Japan have not been established. Although several studies have examined antimicrobial resistance (AMR) in STEC isolated from healthy carriers27–29, comprehensive large-scale analyses combining genomic and phenotypic analyses remain limited.

In this study, to obtain fundamental information relevant to the eradication of STEC from carriers, we analyzed 495 STEC strains previously subjected to whole-genome sequencing from stool-screened individuals, which were classified into more than 100 serotypes, predominantly O156:H25 (13.5%), followed by O174:H21 (7.7%) and O105:H7 (6.1%). We identified antimicrobial resistance genes and conducted antimicrobial susceptibility testing on resistance gene–positive isolates to characterize their resistance profiles.

Materials and methods

Strains used in this study

A total of 495 STEC strains isolated in Japan in 2021 from healthy food handlers and welfare facility workers during routine stool screening were used in this study. These strains were subjected to whole-genome sequencing in a previous study26.

Data analysis

Antimicrobial resistance genes (ARGs) and quinolone resistance–determining region (QRDR) mutations were identified using AMRFinderPlus version 4.0.22 with default settings30. The sequence types (STs) of the strains were identified using SRST2 with default settings31. The plasmid replicon sequences were identified using ABRicate 1.0.1 (https://github.com/tseemann/abricate) with the default settings, employing Plasmidfinder database32. Additional genetic characteristics, including stx subtypes, eae prevalence, and serotype information, were derived from our previously published dataset26.

To construct a core gene–based phylogenetic tree, pangenome analysis was performed using Roary version 3.13.3 with a 90% sequence identity threshold33. Single-nucleotide polymorphism (SNP) sites were extracted from the core gene alignment using SNP-sites version 2.5.1 with the -c option34. A maximum-likelihood phylogenomic tree was subsequently constructed using RAxML-NG version 1.0.1 with the parameters --all, --bs-trees 100, and --model GTR + G435. The resulting tree was visualized and annotated using iTOL version 6.636.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. The antimicrobial agents tested were ampicillin, cefazolin, flomoxef, ceftriaxone, cefozopran, cefmetazole, meropenem, aztreonam, faropenem, sulbactam/cefoperazone, tetracycline, minocycline, gentamicin, amikacin, chloramphenicol, sulfamethoxazole/trimethoprim, nalidixic acid, ciprofloxacin, levofloxacin, fosfomycin, colistin, and azithromycin. Escherichia coli ATCC 25,922 was used as the quality control strain. Susceptibility testing for 19 antimicrobial agents (excluding azithromycin, tetracycline, and nalidixic acid) was outsourced to CRC Co., Ltd. (Kurume, Fukuoka, Japan).

Results

Identification of AMR determinants in STEC from healthy carriers

Genome-based screening for ARGs and QRDR mutations was conducted in 495 STEC strains isolated from asymptomatic carriers. At least one ARG was detected in 83 strains (16.8%), comprising 43 distinct ARGs classified into 11 antimicrobial classes (Fig. 1A and B and Supplemental Table 1). The maximum number of ARGs detected in a single strain was 16 (Fig. 1B). In terms of antimicrobial class, aminoglycoside resistance genes were the most prevalent (66 strains, 13.3%), followed by sulfonamide resistance genes (56 strains, 11.3%) and tetracycline resistance genes (50 strains, 10.1%) (Fig. 1A). In contrast, ARGs associated with colistin (1 strain, 0.2%), rifampicin (2 strains, 0.4%), fosfomycin (5 strains, 1.0%), and macrolides (6 strains, 1.2%) were detected at low frequencies (< 1.5%). QRDR mutations were identified in 3.03% of the strains (Fig. 1A). Overall, 87 strains (17.6%) harbored at least one AMR determinant, defined as either an ARG or a QRDR mutation. Strains carrying resistance determinants from three or more distinct antimicrobial classes, including QRDR mutations (i.e. multidrug-resistant genotypes), accounted for 11.1% of the isolates (Fig. 1C). In addition, 13.9% of the strains carried ARGs belonging to multiple (≥ 2) antimicrobial classes, and 0.8% of the strains harbored ARGs belonging to more than 10 classes.

Fig. 1.

Fig. 1

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Distribution of ARGs and QRDR mutations in 495 STEC strains from asymptomatic carriers, 276 human commensal E. coli strains, and 385 cattle commensal E. coli strains isolated in Japan. Prevalence of ARGs and QRDR mutations by antimicrobial class (A), distribution of the number of ARGs per strain (B), and distribution of ARGs by antimicrobial class (C). Prevalence of individual ARGs and QRDR mutations in STEC from asymptomatic carriers (D), human commensal E. coli (E), and bovine commensal E. coli (F). AG aminoglycosides, TMP trimethoprim, SUL sulfonamide, TET tetracyclines, BL β-lactams, CHL chloramphenicol, MLS macrolide-lincosamide-streptogramin, FOS fosfomycin, RIF rifampicin, COL colistin, QNL quinolone.

Among the 43 ARGs detected, aph(3″)-Ib (11.7%) and aph(6)-Id (11.7%) were the most frequently identified, followed by sul2 (8.7%), tet(A) (7.7%) and blaTEM-1 (6.5%). All the other ARGs were detected at frequencies less than 5%. The extended-spectrum β-lactamase (ESBL) genes blaCTX-M-101 and blaCTX-M-14 were detected in one strain, and blaCTX-M-65 was detected in two strains. Known quinolone resistance–associated QRDR mutations were identified in gyrA, including three mutation patterns: D87N + S83L (0.2%), S83L (1.6%), and D87G (1.0%). In parC, a single mutation pattern (E84V + S80I) was detected in 0.2% of the strains, whereas in parE, one mutation pattern (I529L) was detected in 0.4% of the strains (Fig. 1D).

For comparison, AMR determinants were analyzed under the same criteria in previously reported commensal E. coli isolates from humans (n = 276) and cattle (n = 385) in Japan37. As a result, 34.8% of the human commensal isolates and 38.4% of the cattle commensal isolates harbored at least one ARG (Fig. 1 and Supplemental Table 2). Among the human-derived isolates, aph(3″)-Ib (7.6%), aph(6)-Id (7.6%), sul2 (7.6%), tet(A) (8.3%), and blaTEM-1 (13.7%) were the most prevalent, showing a pattern similar to that observed in STEC from asymptomatic carriers. In contrast, while the prevalence of blaTEM-1 (11.2%) in cattle commensal isolates was comparable to that in human commensal isolates, the frequencies of aph(3″)-Ib (27.8%), aph(6)-Id (27.8%), sul2 (24.9%), tet(A) (15.1%), and tet(B) (14.0%) were substantially greater than those in commensal E. coli, suggesting stronger selective pressure from the corresponding antimicrobial agents in cattle.

Phylogenetic distribution of ARGs among STEC from healthy carriers

Core gene–based phylogenetic analysis revealed that STEC strains isolated from healthy carriers exhibited extensive phylogenetic diversity (Fig. 2). The majority of strains were assigned to phylogroup B1 (80.4%), while the remaining strains belonged to phylogroups A (5.3%), B2 (5.1%), C (4.8%), E (2.8%), G (1.0%), and D (0.6%) (Supplemental Table 1). With respect to toxin profiles, strains harboring stx1 alone, stx2 alone, or both stx1 and stx2 accounted for 40.4%, 49.5%, and 10.1% of the isolates, respectively, and 36.8% of the strains were positive for LEE (Fig. 2 and Supplemental Table 1). The distribution patterns of stx1, stx2, and LEE were heterogeneous even within individual sublineages, suggesting frequent acquisition and loss of these virulence factors through horizontal gene transfer.

Fig. 2.

Fig. 2

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Core gene–based phylogenetic tree of 495 STEC strains isolated from asymptomatic carriers and the distribution of ARGs and QRDR mutations. The maximum-likelihood (ML) tree was constructed on the basis of 189,038 single-nucleotide polymorphism (SNP) sites identified in 2052 core genes and rooted using cryptic Escherichia clade I strain TW15838. Phylogroups, serotypes, and the distributions of stx1, stx2, LEE, ARGs and QRDR mutations are shown below the tree.

Similarly, ARGs showed little phylogenetic clustering and were broadly distributed across the phylogeny, which is consistent with repeated acquisition and loss mediated by mobile genetic elements (Fig. 2). Plasmid replicon typing identified multiple Inc-types widely distributed among isolates, and co-occurrence analysis with resistance genes did not reveal clear associations between specific plasmid types and particular resistance determinants (Supplemental Table 3), indicating that further analyses using long-read sequencing will be required to resolve plasmid contexts.

Strains harboring QRDR mutations also displayed phylogenetic diversity, with identical mutations detected in strains belonging to distinct lineages. Notably, at the serotype level, ARGs were rarely detected in the frequently observed serotypes O156:H25 and O174:H21 (Fig. 2).

Phenotypic AMR profiles of ARGs carrying STEC from healthy carriers

Phenotypic AMR profiles were determined for 87 strains that were positive for at least one AMR gene or QRDR mutation by genome-based analysis using susceptibility testing against 22 antimicrobial agents. Resistant strains were identified for 14 of the tested agents, with tetracycline (n = 50) being the most frequent, followed by ampicillin (n = 33), chloramphenicol (n = 21), and sulfamethoxazole/trimethoprim (n = 17) (Fig. 3 and Supplemental Table 4). No strains exhibited resistance to sulbactam/cefoperazone, cefmetazole, meropenem, faropenem, amikacin, fosfomycin, colistin, or azithromycin. Among the 87 strains tested, 17 were susceptible to all the antimicrobial agents. In addition, 22 strains exhibited resistance to three or more antimicrobial classes and were therefore classified as phenotypically MDR (Supplemental Table 4).

Fig. 3.

Fig. 3

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Concordance between phenotypic and genotypic antimicrobial resistance in STEC isolates from asymptomatic carriers. Among the 495 STEC isolates from asymptomatic carriers, antimicrobial susceptibility testing was performed for 87 isolates that were positive for ARGs and/or QRDR mutations. The corresponding AMR genotypes for each phenotypically resistant isolate are shown. The antimicrobial agents tested were ampicillin (ABPC), cefazolin (CEZ), flomoxef (FMOX), ceftriaxone (CTRX), cefozopran (CZOP), cefmetazole (CMZ), meropenem (MEPM), aztreonam (AZT), faropenem (FRPM), sulbactam/cefoperazone (S/C), tetracycline (TC), minocycline (MINO), gentamicin (GM), amikacin (AMK), chloramphenicol (CP), sulfamethoxazole/trimethoprim (ST), nalidixic acid (NA), ciprofloxacin (CPFX), levofloxacin (LVFX), fosfomycin (FOM), colistin (CL) and azithromycin (AZM).

For all the strains exhibiting phenotypic resistance, corresponding ARGs were detected (Fig. 3). Among the 33 ampicillin-resistant strains, blaTEM-1 was predominant (84.8%). Four strains resistant to cefazolin, flomoxef, ceftriaxone, and cefozopran but susceptible to sulbactam/cefoperazone were classified as ESBL-producing E. coli and harbored blaCTX-M-65 + blaOXA-10 + blaTEM-1 (50%), blaCTX-M-101 (25%), or blaCTX-M-14 (25%); these same strains were also resistant to aztreonam. Among the 50 tetracycline-resistant strains, 72% carried tet(A), and 20% carried tet(B), while five minocycline-resistant strains harbored tet(B) (80%) or tet(B) + tet(M) (20%). Three strains were resistant to gentamicin, and each possessed multiple gentamicin resistance genes. Among the 21 chloramphenicol-resistant strains, floR was most common (52.4%), followed by catA1 (14.3%), with cmlA1, cmlA1 + floR, or cmlA5 + floR each accounting for 9.5%. Seventeen strains were resistant to sulfamethoxazole/trimethoprim and carried diverse gene combinations, including dfrA12 + sul3, dfrA14 + sul2, and dfrA1 + sul1 + sul2 (each 17.6%). Fourteen strains were resistant to nalidixic acid, predominantly harboring gyrA S83L (57.1%) or D87G (35.7%) mutations, whereas one strain resistant to both ciprofloxacin and levofloxacin carried multiple QRDR mutations (gyrA D87N and S83L, parC E84V and S80I, and parE I529L).

Sixty-three strains harboring various combinations of aadA1, aadA2, aadA5, aph(3″)-Ia, aph(3″)-Ib, aph(6)-Id, and sat2 were susceptible to all the aminoglycosides tested in this study (Supplemental Fig. 1). Similarly, six strains carrying lnu(F), mef(B), mef(C) + mph(G), or mph(A) remained susceptible to the tested macrolide, azithromycin. In addition, strains harboring fosA7.7 (n = 5), mcr-3.1 (n = 1), or qnrS1 (n = 7) did not exhibit phenotypic resistance to fosfomycin, colistin, or quinolones, respectively. The strain harboring the parE_I529L mutation alone (n = 1), a QRDR mutation, did not exhibit resistance to quinolones in this study. These findings suggest that the detected ARGs may not increase MICs to levels exceeding the clinical resistance breakpoint under the conditions tested, while the potential loss of ARGs or their associated plasmids during susceptibility testing cannot be ruled out.

Discussion

In this study, we genetically and phenotypically analyzed AMR in 495 STEC isolates from asymptomatic carriers, for which whole-genome sequencing had previously been performed and pathogenic risk had been evaluated through comparison with clinical isolates26. The present analysis aimed to clarify the AMR profiles of these isolates to provide information relevant to potential eradication strategies. Comprehensive identification of ARGs revealed that 83 isolates (16.8%) carried at least one ARG (Fig. 1 and Supplemental Table 1). Genome-based data on ARGs in carrier-derived STEC in Japan remain limited. A prior study of 27 isolates reported a higher ARG prevalence (33.4%)28, but the small sample size may have affected the estimate. Although lower, the prevalence observed here is based on a larger and more representative dataset.

Across the 495 carrier-derived STEC isolates, the most frequently detected ARGs were aph(3″)-Ib (11.7%), aph(6)-Id (11.7%), sul2 (8.7%), tet(A) (7.7%), and blaTEM-1 (6.5%) (Fig. 1). Notably, these same ARGs have been consistently reported at high frequencies in previous studies of both carrier- and patient-derived STEC in Japan28, indicating a stable resistance gene profile across different STEC populations. This consistency suggests that STEC strains persisting in the human intestine may experience antimicrobial selective pressures similar to those acting on commensal E. coli. Outside Japan, AMR in carrier-derived STEC has been reported from China; however, the study included only 10 isolates, limiting direct comparison with the present dataset29. A study from England reported that, among 1,473 E. coli O157 isolates obtained from diarrheal patients, 14.7% carried at least one ARG with aph(3″)-Ib-aph(6)-Id (11.8%), tetA (11.3%), and blaTEM-1 (8.0%) being the most frequently detected genes38. In the United States, analysis of 276 food-derived STEC isolates showed that 33 strains (12%) harbored at least one ARG, most commonly aph(6)-Id (9.4%), sul2 (9.1%), aph(3″)-Ib (8.3%)39. These findings suggest that the overall prevalence of ARG and the predominant resistance genes in STEC in these countries are broadly comparable to those observed in Japan.

We reanalyzed published human- and cattle-derived commensal E. coli genomes using the same criteria. Human isolates showed AMR profiles similar to carrier-derived STEC, whereas cattle isolates harbored ARGs more frequently (Fig. 1 and Supplemental Table 2), suggesting stronger livestock-associated selective pressure.

Phenotypic resistance to tetracycline (n = 50) and ampicillin (n = 33) was most frequently observed, corresponding to 10.1% and 6.7% of all the isolates, respectively (Fig. 3 and Supplemental Table 4). A previous Japanese study examining 279 STEC isolates from healthy food handlers reported high resistance rates to streptomycin (> 34%), ampicillin (> 7.7%), and tetracycline (< 5.1%)27. Although streptomycin susceptibility was not assessed in the present study, the high prevalence of ampicillin and tetracycline resistance is consistent with these earlier findings (Fig. 3 and Supplemental Table 4). Moreover, a genome analysis of 14 MDR isolates in that study revealed aph(6)-Id, aph(3″)-Ib, blaTEM-1B, and tet(A) as the principal resistance determinants27, closely matching the resistance gene distribution observed here (Fig. 1).

aph(6)-Id and aph(3″)-Ib were also frequently detected in the present study (Fig. 1.). These genes are known to confer resistance to streptomycin, suggesting that the strains harboring these genes may also exhibit streptomycin resistance40. These genes do not confer resistance to other aminoglycosides41, such as gentamicin or amikacin, both of which were included in our susceptibility testing and remained effective (Supplemental Fig. 1). Streptomycin is now infrequently used in human clinical practice owing to its adverse effects and the high prevalence of streptomycin-resistant bacteria, and it has largely been replaced by gentamicin and amikacin42. However, streptomycin continues to be used in livestock for the treatment of conditions such as bacterial diarrhea, and high levels of streptomycin resistance have been reported among bacteria of livestock origin43,44. In Japan, streptomycin is also used in companion animal medicine and as an agricultural agent for plant disease control, which may further contribute to environmental selective pressure. Therefore, antimicrobial selective pressure in the livestock, companion animal, and agricultural sectors may indirectly contribute to the persistence and dissemination of streptomycin resistance genes in human-associated E. coli populations.

Known QRDR mutations associated with quinolone resistance were identified in 3.03% of the STEC isolates from asymptomatic carriers (Fig. 1A). However, resistance to fluoroquinolones was observed in only a single strain harboring multiple QRDR mutations (gyrA D87N and S83L, parC E84V and S80I, and parE I529L) (Fig. 3 and Supplemental Table 4). Although fluoroquinolones are known to induce Shiga toxin production in STEC45, previous studies have demonstrated their effectiveness in early-stage STEC infections without inducing HUS, and they are frequently selected for STEC treatment in Japan16.

In the present study, five isolates carried fosA7.5, but no phenotypic resistance to fosfomycin was observed (Fig. 3 and Supplemental Table 4). Certain fosA7.5 variants have been reported to not be associated with an increase in the minimum inhibitory concentration (MIC) beyond the susceptible range in E. coli46. Amikacin has also been recommended in some reports for the eradication of STEC carriage25. No amikacin-resistant isolates were identified, although two isolates showed intermediate susceptibility (Supplemental Table 4). These isolates did not harbor known aminoglycoside resistance genes, suggesting that elevated MICs may be attributable to as-yet-uncharacterized mechanisms. At present, no standardized antimicrobial regimen has been established for the eradication of STEC carriage. Nevertheless, on the basis of the low prevalence of resistance observed in this study, fluoroquinolones, fosfomycin, and amikacin remain potential therapeutic options in Japan.

In conclusion, this study provides a comprehensive genome-based assessment of antimicrobial resistance in STEC isolates from asymptomatic carriers in Japan. Although the overall prevalence of ARGs was limited, resistance determinants were detected across diverse genetic lineages, and their distribution largely mirrored that observed in human commensal E. coli. Phenotypic resistance to ampicillin and tetracycline was most frequently observed, whereas resistance to fluoroquinolones, fosfomycin, and amikacin remained rare. Notably, streptomycin resistance–associated genes were commonly detected, likely reflecting antimicrobial selective pressures beyond the human clinical setting. These findings underscore the importance of continued genomic surveillance of STEC in asymptomatic carriers and highlight the need for integrated monitoring across human and livestock reservoirs to better understand the ecology and transmission dynamics of antimicrobial resistance.

Supplementary Information

Supplementary Information 1. (602.7KB, pdf)
Supplementary Information 2. (125.8KB, xlsx)
Supplementary Information 3. (211.1KB, xlsx)
Supplementary Information 4. (17.1KB, xlsx)
Supplementary Information 5. (35.3KB, xlsx)

Acknowledgements

We thank Ayaka Wakakuwa for providing technical assistance.

Author contributions

YI, TS, TY and YO conceptualized the study. YI, MO, SI, HK, AN, and YO curated the data. YI and TY performed the investigation. TS and YO administered the project. YI and YO wrote the original draft. All the authors were responsible for reviewing and editing the manuscript. All the authors had access to the data presented in this study and had final responsibility for the decision to submit for publication.

Funding

The authors received no financial support for the research, authorship or publication of this article.

Data availability

All genome sequence data analyzed in this study have been deposited in the NCBI BioProject database under the following accession numbers: PRJDB18641 for STEC isolated from asymptomatic carriers, and PRJDB5579 for human- and cattle-derived commensal E. coli.

Competing interests

The authors declare no competing interests.

Consent for Publication

All participants in the study consented to publication of the data obtained in relation to the present study.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Tarr, P. I., Gordon, C. A. & Chandler, W. L. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet365, 1073–1086 (2005). [DOI] [PubMed] [Google Scholar]
  • 2.Kim, J. S., Lee, M. S. & Kim, J. H. Recent updates on outbreaks of Shiga toxin-producing Escherichia coli and its potential reservoirs. Front. Cell. Infect. Microbiol.10, 273 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bai, X., Scheutz, F., Dahlgren, H. M., Hedenstrom, I. & Jernberg, C. Characterization of clinical Escherichia coli strains producing a novel Shiga Toxin 2 subtype in Sweden and Denmark. Microorganisms10.3390/microorganisms9112374 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Gill, A. et al. Characterization of atypical Shiga toxin gene sequences and description of Stx2j, a new subtype. J. Clin. Microbiol.60, e0222921 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Hughes, A. C. et al. Structural and functional characterization of Stx2k, a new subtype of Shiga Toxin 2. Microorganisms10.3390/microorganisms8010004 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Scheutz, F. et al. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J. Clin. Microbiol.50, 2951–2963 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Yang, X. et al. Genomic characterization of Escherichia coli O8 strains producing Shiga Toxin 2l subtype. Microorganisms10.3390/microorganisms10061245 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.De Rauw, K., Buyl, R., Jacquinet, S. & Pierard, D. Risk determinants for the development of typical haemolytic uremic syndrome in Belgium and proposition of a new virulence typing algorithm for Shiga toxin-producing Escherichia coli. Epidemiol. Infect.147, e6 (2018). [DOI] [PubMed] [Google Scholar]
  • 9.Freedman, S. B., van de Kar, N. & Tarr, P. I. Shiga toxin-producing Escherichia coli and the hemolytic-uremic syndrome. N. Engl. J. Med.389, 1402–1414 (2023). [DOI] [PubMed] [Google Scholar]
  • 10.Kalalah, A. A., Koenig, S. S. K., Bono, J. L., Bosilevac, J. M. & Eppinger, M. Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli. Front. Microbiol.15, 1364026 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.McGeoch, L. J. et al. The epidemiology of haemolytic uraemic syndrome in England, 2009-2023: an analysis of national surveillance data. J. Infect.91, 106551 (2025). [DOI] [PubMed] [Google Scholar]
  • 12.Freedman, S. B. et al. Shiga toxin-producing Escherichia coli infection, antibiotics, and risk of developing hemolytic uremic syndrome: a meta-analysis. Clin. Infect. Dis.62, 1251–1258 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Kakoullis, L., Papachristodoulou, E., Chra, P. & Panos, G. Shiga toxin-induced haemolytic uraemic syndrome and the role of antibiotics: a global overview. J. Infect.79, 75–94 (2019). [DOI] [PubMed] [Google Scholar]
  • 14.Ikeda, K. et al. Effect of early fosfomycin treatment on prevention of hemolytic uremic syndrome accompanying Escherichia coli O157:H7 infection. Clin. Nephrol.52, 357–362 (1999). [PubMed] [Google Scholar]
  • 15.Myojin, S. et al. Interventions for Shiga toxin-producing Escherichia coli gastroenteritis and risk of hemolytic uremic syndrome: a population-based matched case control study. PLoS ONE17, e0263349 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Shiomi, M., Togawa, M., Fujita, K. & Murata, R. Effect of early oral fluoroquinolones in hemorrhagic colitis due to Escherichia coli O157:H7. Pediatr. Int.41, 228–232 (1999). [DOI] [PubMed] [Google Scholar]
  • 17.Tajiri, H. et al. A role for fosfomycin treatment in children for prevention of haemolytic-uraemic syndrome accompanying Shiga toxin-producing Escherichia coli infection. Int. J. Antimicrob. Agents46, 586–589 (2015). [DOI] [PubMed] [Google Scholar]
  • 18.Takeda, T. Strategy to prevent the progression of enterohemorrhagic Escherichia coli O157 infection to hemolytic uremic syndrome. Jpn. J. Med. Sci. Biol.51(Suppl), S124-128 (1998). [DOI] [PubMed] [Google Scholar]
  • 19.Tsuboi, I. et al. Antibiotic susceptibility of enterohemorrhagic Escherichia coli O157:H7 isolated from an outbreak in Japan in 1996. Antimicrob. Agents Chemother.42, 431–432 (1998). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Armstrong, G. L., Hollingsworth, J. & Morris, J. G. Jr. Emerging foodborne pathogens: Escherichia coli O157:H7 as a model of entry of a new pathogen into the food supply of the developed world. Epidemiol. Rev.18, 29–51 (1996). [DOI] [PubMed] [Google Scholar]
  • 21.Kintz, E., Brainard, J., Hooper, L. & Hunter, P. Transmission pathways for sporadic Shiga-toxin producing E. coli infections: A systematic review and meta-analysis. Int. J. Hyg. Environ. Health.220, 57–67 (2017). [DOI] [PubMed] [Google Scholar]
  • 22.Terajima, J., Izumiya, H., Hara-Kudo, Y. & Ohnishi, M. Shiga Toxin (Verotoxin)-producing Escherichia coli and foodborne disease: a review. Food Saf (Tokyo)5, 35–53 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Harada, T. et al. Laboratory investigation of an Escherichia coli O157:H7 strain possessing a vtx2c gene with an IS1203 variant insertion sequence isolated from an asymptomatic food handler in Japan. Diagn. Microbiol. Infect. Dis.77, 176–178 (2013). [DOI] [PubMed] [Google Scholar]
  • 24.Terajima, J., Iyoda, S., Ohnishi, M. & Watanabe, H. Shiga toxin (Verotoxin)-producing Escherichia coli in Japan. Microbiol. Spectr.10.1128/microbiolspec.EHEC-0011-2013 (2014). [DOI] [PubMed] [Google Scholar]
  • 25.Sayk, F., Hauswaldt, S., Knobloch, J. K., Rupp, J. & Nitschke, M. Do asymptomatic STEC-long-term carriers need to be isolated or decolonized? New evidence from a community case study and concepts in favor of an individualized strategy. Front. Public Health12, 1364664 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Imai, Y. et al. Genomics of Shiga toxin-producing Escherichia coli from asymptomatic carriers in Japan highlights risk-adaptive control. Commun. Biol.9, 299 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ohata, N., Noda, M., Ohta, K., Hatta, M. & Nakayama, T. Genetic characterization of antimicrobial resistance, virulence, and plasmid-mediated transferability in Enterohaemorrhagic E. coli O157 isolates from healthy food handlers in Japan, 2006–2021. J. Appl. Microbiol.136, lxaf245 (2025). [DOI] [PubMed] [Google Scholar]
  • 28.Baba, H. et al. Genomic analysis of Shiga toxin-producing Escherichia coli from patients and asymptomatic food handlers in Japan. PLoS ONE14, e0225340 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Sui, X. et al. Characteristics of Shiga Toxin-producing Escherichia coli circulating in asymptomatic food handlers. Toxins (Basel)10.3390/toxins15110640 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Feldgarden, M. et al. AMRFinderPlus and the reference gene catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence. Sci. Rep.11, 12728 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Inouye, M. et al. SRST2: rapid genomic surveillance for public health and hospital microbiology labs. Genome Med.6, 90 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Carattoli, A. et al. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob. Agents. Chemother.58, 3895–3903 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Page, A. J. et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics31, 3691–3693 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Page, A. J. et al. SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments. Microb Genom.2, e000056 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Kozlov, A. M., Darriba, D., Flouri, T., Morel, B. & Stamatakis, A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics35, 4453–4455 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res.49, W293–W296 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Arimizu, Y. et al. Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains. Genome Res.29, 1495–1505 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Greig, D. R. et al. Surveillance of antimicrobial resistant Shiga toxin-producing E. coli O157:H7 in England, 2016-2020. J. Antimicrob. Chemother.78, 2263–2273 (2023). [DOI] [PubMed] [Google Scholar]
  • 39.Gonzalez-Escalona, N. & Kase, J. A. Virulence gene profiles and phylogeny of Shiga toxin-positive Escherichia coli strains isolated from FDA regulated foods during 2010-2017. PLoS ONE14, e0214620 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ashenafi, M., Ammosova, T., Nekhai, S. & Byrnes, W. M. Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id, a streptomycin-inactivating enzyme. Mol. Cell. Biochem.387, 207–216 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Wellner, S. M., Alobaidallah, M. S. A., Fei, X., Herrero-Fresno, A. & Olsen, J. E. Genome-wide identification of fitness-genes in aminoglycoside-resistant Escherichia coli during antibiotic stress. Sci. Rep.14, 4163 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Krause, K. M., Serio, A. W., Kane, T. R. & Connolly, L. E. Aminoglycosides: an overview. Cold Spring Harb. Perspect. Med.6, a027029 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Fujimoto, H., Nakamura, T., Sato, A. & Chuma, T. Antimicrobial susceptibility of Trueperella pyogenes isolated from cattle and pigs with septicemia in southern Kyushu, Japan. J. Vet. Med. Sci.85, 379–382 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Sasaki, Y. et al. Antimicrobial resistance of Salmonella isolates from cattle and horses with salmonellosis in Hokkaido, Japan. J. Vet. Med. Sci.86, 1227–1232 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Kimmitt, P. T., Harwood, C. R. & Barer, M. R. Induction of type 2 Shiga toxin synthesis in Escherichia coli O157 by 4-quinolones. Lancet353, 1588–1589 (1999). [DOI] [PubMed] [Google Scholar]
  • 46.Milner, K. A. et al. Identification and characterization of a novel FosA7 member from Fosfomycin-resistant Escherichia coli clinical isolates from Canadian hospitals. Antimicrob. Agents Chemother.10.1128/AAC.00865-20 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information 1. (602.7KB, pdf)
Supplementary Information 2. (125.8KB, xlsx)
Supplementary Information 3. (211.1KB, xlsx)
Supplementary Information 4. (17.1KB, xlsx)
Supplementary Information 5. (35.3KB, xlsx)

Data Availability Statement

All genome sequence data analyzed in this study have been deposited in the NCBI BioProject database under the following accession numbers: PRJDB18641 for STEC isolated from asymptomatic carriers, and PRJDB5579 for human- and cattle-derived commensal E. coli.

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