Antimicrobial-Resistant Enteric Gram-Negative Bacteria Isolated from a Fatal Diarrhea in a Horse: Genomic Characterization of CTX-M-2-Producing Escherichia coli.
Abstract: Infections caused by antimicrobial-resistant bacteria are difficult to treat and increase the risk of death in animals. This report describes a fatal case of diarrhea in a horse that, despite intensive treatment including surgery and broad-spectrum antimicrobials (ceftiofur and amikacin), experienced a worsening of its condition and subsequent death. A fecal swab sample was subjected to microbiological culture for the identification of bacteria and assessment of their phenotypical antimicrobial susceptibility profiles using the disk-diffusion and broth microdilution methods. The double-disk synergy test, polymerase chain reactions for the detection of genes encoding extended-spectrum β-lactamases, and whole-genome sequence-based analysis were also performed. Strains of and were isolated, with the strain DSL-HVUVV-2025 presenting resistance to a third-generation cephalosporin. Accordingly, the gene was identified in the DSL-HVUVV-2025 strain, which was submitted to whole-genome sequencing. Genomic analysis showed several antimicrobial resistance determinants, as well as virulence genes, including those associated with the enteroaggregative pathotype. The gene was surrounded by an IS element and embedded in a complex class 1 integron that is part of the Tn transposon. Strain DSL-HVUVV-2025 belonged to a novel sequence type. This case highlights the importance of monitoring antimicrobial resistance and performing genomic characterization of bacteria involved in equine diarrhea to guide effective clinical management in veterinary hospitals. It also reinforces the role of horses as potential carriers of WHO critical priority pathogens and the need for responsible antimicrobial use.
Publication Date: 2025-11-21 PubMed ID: 41463689PubMed Central: PMC12729381DOI: 10.3390/antibiotics14121185Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
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Summary
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Antimicrobial-resistant bacteria caused a fatal diarrheal infection in a horse, and detailed genetic analysis identified a specific resistant Escherichia coli strain carrying CTX-M-2, highlighting the importance of monitoring and responsible antibiotic use in veterinary care.
Study Background and Objective
- Infections by bacteria resistant to antimicrobial drugs pose treatment challenges and increase mortality in animals.
- The study focuses on a fatal case of diarrhea in a horse that did not improve despite intensive treatment including surgery and broad-spectrum antibiotics (ceftiofur and amikacin).
- The main goal was to identify the bacterial strains involved, characterize their antimicrobial resistance, and provide genomic insights into resistance mechanisms.
Methods Used in the Study
- A fecal swab from the affected horse was collected for microbiological culture to isolate bacteria.
- Phenotypical antimicrobial susceptibility testing was performed using:
- Disk-diffusion method
- Broth microdilution method
- Double-disk synergy test was conducted to detect extended-spectrum β-lactamase (ESBL) production.
- Polymerase chain reaction (PCR) was used to detect genes that encode ESBLs, focusing on the blaCTX-M-2 gene.
- Whole-genome sequencing (WGS) was performed on the Escherichia coli strain to analyze resistance genes and virulence factors.
Key Findings
- Multiple enteric Gram-negative bacteria strains were isolated; notable was the Escherichia coli strain DSL-HVUVV-2025.
- This E. coli strain showed resistance to third-generation cephalosporins, important antibiotics in veterinary and human medicine.
- The blaCTX-M-2 gene responsible for ESBL production was identified in this strain.
- Genomic analysis revealed:
- Multiple antimicrobial resistance genes implying multidrug resistance capabilities.
- Virulence genes associated with the enteroaggregative E. coli pathotype.
- The blaCTX-M-2 gene was located within a complex class 1 integron, which itself is part of a Tn transposon, indicating mobile genetic elements that facilitate spreading of resistance.
- An insertion sequence (IS) element was surrounding the resistance gene, further highlighting the potential for gene mobility.
- The strain belonged to a novel sequence type, suggesting it was a previously uncharacterized lineage.
Implications and Conclusions
- This fatal case emphasizes the critical need for continuous surveillance of antimicrobial resistance in bacteria involved in equine infections.
- Genomic characterization aids in understanding resistance mechanisms and guiding effective clinical management in veterinary settings.
- Horses may serve as reservoirs or carriers of WHO critical priority pathogens, including multidrug-resistant bacteria.
- There is a strong call for responsible antimicrobial use in veterinary medicine to prevent the emergence and spread of resistant pathogens.
- The findings highlight the One Health concern, where antimicrobial resistance in animals can impact human and environmental health.
Cite This Article
APA
Rossi GAM, Sellera FP, Ferraz CM, Carvalho RS, Oliveira APL, Marques CA, Fávaro EBR, Rosa RDS, Silva LAM, Cardozo MV, Stehling EG, Furlan JPR.
(2025).
Antimicrobial-Resistant Enteric Gram-Negative Bacteria Isolated from a Fatal Diarrhea in a Horse: Genomic Characterization of CTX-M-2-Producing Escherichia coli.
Antibiotics (Basel), 14(12), 1185.
https://doi.org/10.3390/antibiotics14121185 Publication
Researcher Affiliations
- Department of Veterinary Medicine, University Vila Velha (UVV), Av. Comissário José Dantas de Melo, n.21, Vila Velha 29102-920, ES, Brazil.
- School of Veterinary Medicine, Metropolitan University of Santos, Santos 11045-002, SP, Brazil.
- Department of Internal Medicine, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo 05508-220, SP, Brazil.
- Department of Veterinary Medicine, University Vila Velha (UVV), Av. Comissário José Dantas de Melo, n.21, Vila Velha 29102-920, ES, Brazil.
- Department of Veterinary Medicine, University Vila Velha (UVV), Av. Comissário José Dantas de Melo, n.21, Vila Velha 29102-920, ES, Brazil.
- Department of Veterinary Medicine, University Vila Velha (UVV), Av. Comissário José Dantas de Melo, n.21, Vila Velha 29102-920, ES, Brazil.
- Department of Veterinary Medicine, University Vila Velha (UVV), Av. Comissário José Dantas de Melo, n.21, Vila Velha 29102-920, ES, Brazil.
- Department of Veterinary Medicine, University Vila Velha (UVV), Av. Comissário José Dantas de Melo, n.21, Vila Velha 29102-920, ES, Brazil.
- Department of Clinical Analyses, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto 19040-903, SP, Brazil.
- Department of Pathology, Reproduction and One Health, Faculty of Agricultural and Veterinary Sciences, São Paulo State University (UNESP), Jaboticabal 14884-900, SP, Brazil.
- Department of Pathology, Reproduction and One Health, Faculty of Agricultural and Veterinary Sciences, São Paulo State University (UNESP), Jaboticabal 14884-900, SP, Brazil.
- Department of Clinical Analyses, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto 19040-903, SP, Brazil.
- Department of Pharmaceutical Sciences, Health Sciences Center, Federal University of Paraíba, João Pessoa 58051-900, PB, Brazil.
Grant Funding
- 139/2021 2021-WTG95 / FAPES
- 23/12947-4 / FAPESP
- 551/2023 P 2023-RH7P2 / FAPES
- 304905/2022-4 / CNPq
- 88887.824722/2023-00 / CAPES
- Financial Code 001 / CAPES
Conflict of Interest Statement
The authors declare no conflicts of interest.
References
This article includes 47 references
- Uzal FA, Arroyo LG, Navarro MA, Gomez DE, Asín J, Henderson EB. Bacterial and viral enterocolitis in horses: A review.. J. Vet. Diagn. Investig. 2022;34:354–375.
- Shaw SD, Stampfli H. Diagnosis and treatment of undifferentiated and infectious acute diarrhea in the adult horse.. Vet. Clin. N. Am. Equine Pract. 2018;34:39–53.
- Cella E, Giovanetti M, Benedetti F, Scarpa F, Johnston C, Borsetti A, Ceccarelli G, Azarian T, Zella D, Ciccozzi M. Joining forces against antibiotic resistance: The One Health solution.. Pathogens 2023;12:1074.
- Macesic N, Uhlemann AC, Peleg AY. Multidrug-resistant Gram-negative bacterial infections.. Lancet 2025;405:257–272.
- Partridge SR, Kwong SM, Firth N, Jensen SO. Mobile genetic elements associated with antimicrobial resistance.. Clin. Microbiol. Rev. 2018;31:e00088-17.
- Pornsukarom S, Thakur S. Horizontal dissemination of antimicrobial resistance determinants in multiple Salmonella serotypes following isolation from the commercial swine operation environment after manure application.. Appl. Environ. Microbiol. 2017;83:e01503-17.
- Poirel L, Madec JY, Lupo A, Schink AK, Kieffer N, Nordmann P, Schwarz S. Antimicrobial resistance in Escherichia coli.. Microbiol. Spectr. 2018;6:10.1128/microbiolspec.arba-0026-2017.
- Wójcicki M, Chmielarczyk A, Świder O, Średnicka P, Strus M, Kasperski T, Shymialevich D, Cieślak H, Emanowicz P, Kowalczyk M. Bacterial pathogens in the food industry: Antibiotic resistance and virulence factors of Salmonella enterica strains isolated from food chain links.. Pathogens 2022;11:1323.
- Maddox TW, Clegg PD, Williams NJ, Pinchbeck GL. Antimicrobial resistance in bacteria from horses: Epidemiology of antimicrobial resistance.. Equine Vet. J. 2015;47:756–765.
- Awosile BB, Heider LC, Saab ME, McClure JT. Antimicrobial resistance in bacteria isolated from horses from the Atlantic Provinces, Canada (1994 to 2013). Can. Vet. J. 2018;59:951–957.
- Pimenta J, Pinto AR, Saavedra MJ, Cotovio M. Equine Gram-negative oral microbiota: An antimicrobial resistances watcher?. Antibiotics 2023;12:792.
- Clayton MH. Conditioning Sport Horses.. .
- Casemiro PAF, Ferraz CM, Reis MS, Menezes MP, Tobias FL, Rossi GAM, Cardozo MV. Retrospective study on antimicrobial resistance in bacteria isolated from clinical infections in Dogs at a Brazilian Veterinary Teaching Hospital.. Top. Companion Anim. Med. 2025;68:101004.
- Drzewiecka D. Significance and roles of Proteus spp. bacteria in natural environments.. Microb. Ecol. 2016;72:741–758.
- . Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals.. 2023.
- BrCAST. BrCAST; Rio de Janeiro, Brazil: 2024. Version 14.0; Brazilian Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters.
- Jesudason T. WHO publishes updated list of bacterial priority pathogens. Lancet Microbe 2024;5:100940.
- Drieux L, Brossier F, Sougakoff W, Jarlier V. Phenotypic detection of extended-spectrum beta-lactamase production in Enterobacteriaceae: Review and bench guide. Clin. Microbiol. Infect. 2008;14((Suppl. S1)):90–103.
- Dallenne C, Da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother. 2010;65:490–495.
- Zhou Z, Alikhan N.F, Mohamed K, Fan Y, Achtman A, Agama Study Group. The EnteroBase user’s guide, with case studies on Salmonella transmissions, Yersinia pestis phylogeny, and Escherichia core genomic diversity. Genome Res. 2020;30:138–152.
- Kabir A, Lamichhane B, Habib T, Adams A, El-Sheikh Ali H, Slovis N.M, Troedsson M.H.T, Helmy Y.A. Antimicrobial Resistance in Equines: A Growing Threat to Horse Health and Beyond—A Comprehensive Review. Antibiotics 2024;13:713.
- Ryan C.A, McNeal C.D, Credille B.C. Ceftiofur use and antimicrobial stewardship in the horse. Equine Vet. J. 2023;55:944–961.
- Redpath A, Hallowell G.D, Bowen I.M. Use of aminoglycoside antibiotics in equine clinical practice; a questionnaire-based study of current use. Vet. Med. Sci. 2020;7:279–288.
- Braz V.S, Melchior K, Moreira C.G. Escherichia coli as a multifaceted pathogenic and versatile bacterium. Front. Cell. Infect. Microbiol. 2020;10:548492.
- Gomes T.A, Elias W.P, Scaletsky I.C, Guth B.E, Rodrigues J.F, Piazza R.M, Ferreira L.C, Martinez M.B. Diarrheagenic Escherichia coli. Braz. J. Microbiol. 2016;47((Suppl. S1)):3–30.
- Samir A, Abdel-Moein K.A, Zaher H.M. Predominance of enterotoxigenic Escherichia coli among ESBL/plasmid-mediated AmpC-producing strains isolated from diarrheic foals: A public health concern. Acta Vet. Scand. 2024;66:54.
- Park T, Cheong H, Yoon J, Kim A, Yun Y, Unno T. Comparison of the Fecal Microbiota of Horses with Intestinal Disease and Their Healthy Counterparts. Vet. Sci. 2021;8:113.
- Dias R.C.B, Tanabe R.H.S, Vieira M.A, Cergole-Novella M.C, Santos L.F.D, Gomes T.A.T, Elias W.P, Hernandes R.T. Analysis of the Virulence Profile and Phenotypic Features of Typical and Atypical Enteroaggregative Escherichia coli (EAEC) Isolated From Diarrheal Patients in Brazil. Front. Cell. Infect. Microbiol. 2020;10:144.
- Luo Q, Kumar P, Vickers T.J, Sheikh A, Lewis W.G, Rasko D.A, Sistrunk J, Fleckenstein J.M. Enterotoxigenic Escherichia coli secretes a highly conserved mucin-degrading metalloprotease to effectively engage intestinal epithelial cells. Infect. Immun. 2014;82:509–521.
- van Duijkeren E, van Asten A.J.A.M, Gaastra W. Chacracterization of Escherichia coli isolated from adult horses with and without enteritis. Vet. Q. 2000;22:162–166.
- James E.M, Kimera Z.I, Mgaya F.X, Niccodem E.M, Efraim J.E, Matee M.I, Mbugi E.V. Occurrence of virulence genes in multidrug-resistant Escherichia coli isolates from humans, animals, and the environment: One Health perspective. PLoS ONE 2025;20:e0317874.
- Guzmán-Blanco M, Labarca J.A, Villegas M.V, Gotuzzo E, Latin America Working Group on Bacterial Resistance. Extended spectrum β-lactamase producers among nosocomial Enterobacteriaceae in Latin America. Braz. J. Infect. Dis. 2014;18:421–433.
- Han J, Aljahdali N, Zhao S, Tang H, Harbottle H, Hoffmann M, Frye J.G, Foley S.L. Infection biology of Salmonella enterica. EcoSal Plus 2024;12:eesp-0001-2023.
- Kabir A, Kelley W.G, Glover C, Erol E, Helmy Y.A. Phenotypic and genotypic characterization of antimicrobial resistance and virulence profiles of Salmonella enterica serotypes isolated from necropsied horses in Kentucky. Microbiol. Spectr. 2025;13:e02501-24.
- Bolzani L, Canter M, Lamperti L, Scaltriti E, Morganti M, Poeta A, Vecchi M, Paglioli S, Rampini A, Ramoni P. Salmonella in horses at slaughter and public health effects in Italy. Int. J. Food Microbiol. 2024;408:110429.
- Rocha F.R, Pinto V.P, Barbosa F.C. The Spread of CTX-M-Type Extended-Spectrum β-Lactamases in Brazil: A Systematic Review. Microb. Drug Resist. 2016;22:301–311.
- Isgren C.M, Edwards T, Pinchbeck G.L, Winward E, Adams E.R, Norton P, Timofte D, Maddox T.W, Clegg P.D, Williams N.J. Emergence of carriage of CTX-M-15 in faecal Escherichia coli in horses at an equine hospital in the UK; increasing prevalence of over a decade (2008–2017). BMC Vet. Res. 2019;15:268.
- Yiğin A. Antimicrobial resistance and extended-spectrum beta-lactamase (ESBL) genes in E. coli isolated from equine fecal samples in Turkey. J. Equine Vet. Sci. 2021;101:103461.
- Adams R.J, Mollenkopf D.F, Mathys D.A, Whittle A, Ballash G.A, Mudge M, Daniels J.B, Barr B, Wittum T.E. Prevalence of extended-spectrum cephalosporin-, carbapenem-, and fluoroquinolone-resistant members of the family Enterobacteriaceae isolated from the feces of horses and hospital surfaces at two equine specialty hospitals. J. Am. Vet. Med. Assoc. 2021;258:758–766.
- Kauter A, Epping L, Ghazisaeedi F, Lubke-Becker A, Wolf S.A, Kannapin D, Stoeckle S.D, Semmler T, Gunther S, Gehlen H. Frequency, local dynamics, and genomic characteristics of ESBL-Producing Escherichia coli isolated from specimens of hospitalized horses. Front. Microbiol. 2021;12:671676.
- Eskola K, Aimo-Koivisto E, Heikinhemo A, Mykkanen A, Hautajarvi T, Gronthal T. Prevalence, risk factors, and characterisation of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E) in horses entering an equine hospital and description of longitudinal excretion. BMC Vet. Res. 2024;20:412.
- Leigue L, Warth J.F.G, Melo L.C, Silva K.C, Moura R.A, Barbato L, Silva L.C, Santos A.C.M, Silva R.M, Lincopan N. MDR ST2179-CTX-M-15 Escherichia coli co-producing RmtD and AAC(6´)-Ib-cr in a horse with extraintestinal infection, Brazil. J. Antimicrob. Chemother. 2014;70:1263–1265.
- Fernandes M.R, Cerdeira L, Silva M.M, Sellera F.P, Muñoz M, Junior F.G, Azevedo S.S, Power P, Gutkind G, Lincopan N. Novel mcr-5.3 variant in a CTX-M-8-producing Escherichia coli ST711 isolated from an infected horse. J. Antimicrob. Chemother. 2018;73:3520–3522.
- Elias L, Gillis D.C, Gurrola-Rodriguez T, Jeon J.H, Lee J.H, Kim T.Y, Lee S.H, Murray S.A, Ohta N, Scott H.M. The Occurrence and Characterization of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolated from Clinical Diagnostic Specimens of Equine Origin. Animals 2020;10:28.
- Smet A, Boyen F, Flahou B, Doublet B, Praud K, Martens A, Butaye P, Cloeckaert A, Haesebrouck F. Emergence of CTX-M-2-producing Escherichia coli in diseased horses: Evidence of genetic exchanges of blaCTX-M-2 linked to ISCR1. J. Antimicrob. Chemother. 2012;67:1289–1291.
- Thomson K, Eskola K, Eklund M, Suominen K, Maatta M, Junnila J, Nykasenoja S, Niinisto K, Gronthal T, Rantala M. Characterisation of and risk factors for extended-spectrum β-lactamase producing Enterobacterales (ESBL-E) in an equine hospital with a special reference to an outbreak caused by Klebsiella pneumoniae ST307:CTX-M-1. Acta Vet. Scand. 2022;64:4.
- Shnaiderman-Torban A, Navon-Venezia S, Dor Z, Paitan Y, Arielly H, Ahmad W.A, Kelmer G, Fulde M, Steinman A. Extended-Spectrum β-lactamase-Producing Enterobacteriaceae Shedding in Farm Horses Versus Hospitalized Horses: Prevalence and Risk Factors. Animals 2020;10:282.
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