Analyze Diet
Veterinary research communications2025; 50(2); 83; doi: 10.1007/s11259-025-11037-8

Characterization of Staphylococci colonizing healthy equine skin: antibiotic resistance, virulence factors, and biofilm formation.

Abstract: In order to develop non-antibiotic therapies to treat dermatological diseases it is urgent to spread knowledge on composition and properties of skin bacteria in healthy animals. Since horses are popular companions of humans, it is necessary to know what risk skin bacteria pose to humans. Therefore the aim of this work was to analyse species composition of staphylococci isolated from skin swabs of 50 healthy horses using MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) spectrometry and to characterize their virulence properties. Swabs were collected from five body areas (neck, dorsal back, abdomen, pastern and muzzle). Among 19 different staphylococcal species identified, the most common species were Staphylococcus succinus, S. xylosus, S. vitulinus and S. warneri. The most frequent antimicrobial resistance in staphylococcal isolates was observed for penicillin G (48%). The blaZ gene was detected in 90% of penicillin resistant isolates while mecA and mecC genes were not observed. Only low level resistance was noted for erythromycin (12%), tetracycline (6%), linezolid (4%) and oxacillin (4%). No multidrug-resistant strain was found, the MAR (multiple antibiotic resistant) index in average was 0.048 ± 0.039. Biofilm production was observed in 90.1% of isolates whereas 62% of them showed strong production. Gelatinase, DNase, protease and lipase activity was found in 50.4%, 41.3%, 22.3% and 46.3% of isolates, respectively. The results of enzyme activities testing using API ZYM kits (BioMérieux) revealed common production of acid and alkaline phosphatase, esterase, esterase lipase and napthtol-AS-BI-phosphohydrolase. The caution when interacting with horses is important especially in the case of injury since their staphylococci showed many virulent characteristics however they were not multi-resistant.
Publication Date: 2025-12-23 PubMed ID: 41432965PubMed Central: 11938290DOI: 10.1007/s11259-025-11037-8Google 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.
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study analyzed the types of staphylococcal bacteria present on the skin of healthy horses and examined their antibiotic resistance, virulence factors, and ability to form biofilms, to assess potential risks to horse and human health.
  • The research aimed to better understand these bacteria to inform non-antibiotic treatments for horse skin diseases and evaluate any zoonotic risk posed to humans.

Introduction and Purpose

  • Horses are common companion animals, making the characterization of their skin microbiota important for both veterinary and human health.
  • Understanding the species composition and properties of staphylococci on healthy equine skin helps in developing alternative therapies to antibiotics.
  • The study specifically focused on assessing antibiotic resistance, virulence factors such as enzyme activity, and biofilm-forming ability of these bacteria.

Methods

  • Skin swabs were collected from five distinct body areas of 50 healthy horses: neck, dorsal back, abdomen, pastern (lower leg), and muzzle.
  • Identification of staphylococcal species was performed using MALDI-TOF mass spectrometry, a rapid and accurate technique for bacterial species determination.
  • Antibiotic resistance profiles were examined by testing sensitivity to commonly used antibiotics and detecting resistance genes such as blaZ, mecA, and mecC.
  • Biofilm formation was assessed, as biofilms contribute to bacterial virulence and antibiotic tolerance.
  • Virulence factors were further evaluated by measuring enzyme activities, including gelatinase, DNase, protease, lipase, and others, using API ZYM kits.

Key Findings – Species Diversity

  • A total of 19 different staphylococcal species were identified, indicating a diverse skin microbiota on horses.
  • The most common species isolated were:
    • Staphylococcus succinus
    • Staphylococcus xylosus
    • Staphylococcus vitulinus
    • Staphylococcus warneri

Antibiotic Resistance

  • Nearly half (48%) of the staphylococcal isolates displayed resistance to penicillin G.
  • The blaZ gene, responsible for beta-lactam resistance, was present in 90% of those penicillin-resistant strains.
  • Resistance genes mecA and mecC, typically associated with methicillin resistance, were not found in any isolates.
  • Low levels of resistance were detected for other antibiotics:
    • Erythromycin: 12%
    • Tetracycline: 6%
    • Linezolid: 4%
    • Oxacillin: 4%
  • No multidrug-resistant staphylococcal strains were identified.
  • The Multiple Antibiotic Resistant (MAR) index was low on average (0.048 ± 0.039), indicating limited exposure to antibiotics or resistance pressure.

Virulence Factors and Biofilm Formation

  • A high proportion (90.1%) of isolates produced biofilms, with 62% showing strong biofilm production, which can protect bacteria from the host immune system and antibiotics.
  • Enzyme activities detected among the isolates included:
    • Gelatinase: 50.4% of isolates
    • DNase: 41.3%
    • Protease: 22.3%
    • Lipase: 46.3%
  • The API ZYM enzymatic testing further revealed frequent production of:
    • Acid and alkaline phosphatases
    • Esterase and esterase lipase
    • Napththol-AS-BI-phosphohydrolase

Implications and Conclusions

  • The skin of healthy horses harbors a variety of staphylococcal species with several virulence factors, indicating these bacteria have potential to cause infection if the skin barrier is breached.
  • The presence of virulent bacteria underlines the importance of caution when handling horses, especially in situations where injuries could allow bacterial entry.
  • Although virulence traits are common, the lack of multidrug resistance suggests that current antibiotic therapies remain effective against these skin bacteria.
  • This study’s findings provide crucial knowledge towards developing alternative, non-antibiotic treatments for equine skin diseases and assessing zoonotic risks.

Cite This Article

APA
Strompfová V, Štempelová L, Bujňáková D, Karahutová L, Gondoľová D, Nagyová M, Siegfried L. (2025). Characterization of Staphylococci colonizing healthy equine skin: antibiotic resistance, virulence factors, and biofilm formation. Vet Res Commun, 50(2), 83. https://doi.org/10.1007/s11259-025-11037-8

Publication

ISSN: 1573-7446
NlmUniqueID: 8100520
Country: Switzerland
Language: English
Volume: 50
Issue: 2
Pages: 83

Researcher Affiliations

Strompfová, Viola
  • Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology, Šoltésovej 4-6, Košice, 040 01, Slovakia.
Štempelová, Lucia
  • Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology, Šoltésovej 4-6, Košice, 040 01, Slovakia. stempelova@saske.sk.
Bujňáková, Dobroslava
  • Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology, Šoltésovej 4-6, Košice, 040 01, Slovakia.
Karahutová, Lívia
  • Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology, Šoltésovej 4-6, Košice, 040 01, Slovakia.
Gondoľová, Dajana
  • Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology, Šoltésovej 4-6, Košice, 040 01, Slovakia.
  • University of Veterinary Medicine and Pharmacy, Komenského 73, Košice, 041 81, Slovakia.
Nagyová, Mária
  • Faculty of Medicine, Department of Medical and Clinical Microbiology, University of P. J. Šafárik in Košice, Trieda SNP 1, Košice, 040 11, Slovakia.
Siegfried, Leonard
  • Faculty of Medicine, Department of Medical and Clinical Microbiology, University of P. J. Šafárik in Košice, Trieda SNP 1, Košice, 040 11, Slovakia.

MeSH Terms

  • Animals
  • Horses / microbiology
  • Biofilms / growth & development
  • Staphylococcus / drug effects
  • Staphylococcus / physiology
  • Staphylococcus / genetics
  • Staphylococcus / pathogenicity
  • Virulence Factors / genetics
  • Virulence Factors / metabolism
  • Skin / microbiology
  • Anti-Bacterial Agents / pharmacology
  • Drug Resistance, Bacterial
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / veterinary

Conflict of Interest Statement

Declarations. Ethics approval: The study involved horses for one-time collection of superficial swab samples. All manipulation was in accordance with standard veterinary practices according Slovak legislation (no. 377/2012 and 436/2012). Competing interests: The authors declare no competing interests.

References

This article includes 39 references
  1. Allano M, Arsenault J, Archambault M, Fairbrother J-H, Sauvé F. Prevalence and risk factors of Staphylococcus aureus nasal colonization in horses admitted to a veterinary teaching hospital. J Vet Intern Med 39:e70027.
    doi: 10.1111/jvim.70027pubmed: 40135807pmc: 11938290google scholar: lookup
  2. Amoako KK, Goto Y, Misawa N, Xu DL, Shinjo T. The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate. FEMS Microbiol Lett 168:65–70.
  3. Arciola CR, Campoccia D, Gamberini S, Carvellati M, Donati E, Montanaro E. Detection of slime production by means of an optimised congo red agar plate test based on a colourimetric scale in Staphylococcus epidermidis clinical isolates genotyped for Ica locus. Biomaterials 23:4233–4239.
    doi: 10.1016/s0142-9612(02)00171-0pubmed: 12194526google scholar: lookup
  4. Baca OG, Roman MJ, Glew RH, Christner RF, Buhler JE, Aragon AS. Acid phosphatase activity in Coxiella burnetii: a possible virulence factor. Infect Immun 61:4232–4239.
    doi: 10.1128/iai.61.10.4232-4239.1993pubmed: 8406811pmc: 281149google scholar: lookup
  5. Bertelloni F, Cagnoli G, Ebani VV. Virulence and antimicrobial resistance in canine Staphylococcus spp. Isolates. Microorganisms 9:515.
    doi: 10.3390/microorganisms9030515pubmed: 33801518pmc: 7998746google scholar: lookup
  6. Bessede E, Angla-gre M, Delagarde Y, Sep Hieng S, Ménard A, Mégraud F. Matrix-assisted laser-desorption/ionization biotyper: experience in the routine of a university hospital. Clin Microbiol Infect 17:533–538.
  7. Bujňáková D, Kmeť V. Functional properties of Lactobacillus strains isolated from dairy products. Folia Microbiol 57:263–267.
    doi: 10.1007/s12223-012-0121-xgoogle scholar: lookup
  8. Clinical and Laboratory Standards Institute. M100 performance standards for antimicrobial susceptibility testing. 30th edition. pp 58–66.
  9. Cuny C, Layer F, Strommenger B, Witte W. Rare occurrence of methicillin-resistant Staphylococcus aureus cc130 with a novel Meca homologue in humans in Germany. PLoS ONE 6:e24360.
    doi: 10.1371/journal.pone.0024360pubmed: 21931689pmc: 3169590google scholar: lookup
  10. Danikowski KM, Cheng T. Alkaline phosphatase activity of Staphylococcus aureus grownin biofilm and suspension cultures. Curr Microbiol 75:1226–1230.
    doi: 10.1007/s00284-018-1514-0pubmed: 29846772google scholar: lookup
  11. Grice EA, Segre JA. The skin Microbiome. Nat Rev Microbiol 9:244–253.
    doi: 10.1038/nrmicro2537pubmed: 21407241pmc: 3535073google scholar: lookup
  12. Harris-Tryon TA, Grice EA. Microbiota and maintenance of skin barrier function. Sci 376:940–945.
    doi: 10.1126/science.abo0693google scholar: lookup
  13. Khusro A, Aarti C, Barbabosa-Pilego A, Hernández SR. Anti-pathogenic, antibiofilm, and technological properties of fermented food associated Staphylococcus succinus strain AAS2. Prepar Biochem Biotechnol 49:176–183.
  14. Krumperman PH. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol 46:165–170.
    doi: 10.1128/aem.46.1.165-170.1983pubmed: 6351743pmc: 239283google scholar: lookup
  15. Kumar D, Kumar L, Nagar S, Raina C, Parshad R, Gupta VK. Screening, isolation and production of lipase/esterase producing Bacillus sp. strain DVL2 and its potential evaluation in esterification and resolution reactions. Arch Appl Sci Res 4:1763–1770.
  16. Leclercq R, Courvalin P. Bacterial resistance to macrolide, lincosamide, and Streptogramin antibiotics by target modification. Antimicrob Agents Chemother 1991 35:1267–1272.
    doi: 10.1128/AAC.35.7.1273pubmed: 1929280pmc: 245156google scholar: lookup
  17. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monne DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012 18:268–281.
  18. Martineau F, Picard FJ, Lansac N, Ménard C, Roy PH, Ouellette M, Bergeron MG. Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother 2000 44:231–238.
    doi: 10.1128/AAC.44.2.231-238.2000pubmed: 10639342pmc: 89663google scholar: lookup
  19. Mick V, Domínguez MA, Tubau F, Liñares J, Pujol M, Martín R. Molecular characterization of resistance to rifampicin in an emerging hospital-associated Methicillin-resistant Staphylococcus aureus clone ST228, Spain. BMC Microbiol 2010 10:68.
    doi: 10.1186/1471-2180-10-68pubmed: 20202188pmc: 2844403google scholar: lookup
  20. Moriello KA. Structure of the skin in horses. MSD Man .
  21. Mota SL, dos Santos LO, Vidaletti MR, Rodrigues RO, Coppola MD, Mayer FQ. Antimicrobial resistance of Coagulase-positive Staphylococcus isolated from healthy Crioulo horses and associated risk factors. J Equine Vet Sci 2021 107:103779.
    doi: 10.1016/j.jevs.2021.103779pubmed: 34802621google scholar: lookup
  22. Nagase N, Sasaki A, Yamashita K, Shimizu A, Wakita Y, Kitai S, Kawano J. Isolation and species distribution of Staphylococci from animal and human skin. J Vet Med Sci 2002 64:245–250.
    doi: 10.1292/jvms.64.245pubmed: 11999444google scholar: lookup
  23. Ng L-K, Martin I, Alfa M, Mulvey M. Multiplex PCR for the detection of Tetracycline resistant genes. Molecul Cellul Probes 2001 15:209–215.
    doi: 10.1006/mcpr.2001.0363google scholar: lookup
  24. Nováková D, Sedlácek I, Pantůcek R, Stetina V, Svec P, Petrás P. Staphylococcus equorum and Staphylococcus succinus isolated from human clinical specimens. J Med Microbiol 2006 55:523–528.
    doi: 10.1099/jmm.0.46246-0pubmed: 16585638google scholar: lookup
  25. O’Shaughnessy-Hunter LC, Yu A, Rousseau JD, Foster RA, Weese JS. Longitudinal study of the cutaneous microbiota of healthy horses. Vet Dermatol 2021 32:467–e128.
    doi: 10.1111/vde.12983pubmed: 34165828google scholar: lookup
  26. Olivo G, Zakia LS, Ribeiro MG, Cunha MLRS, Riboli DFM, Mello PL, Teixeira NB, Araújo CET, Oliveira-Filho JP, Borges AS. Methicillin-resistant Staphylococcus spp. Investigation in hospitalized horses and contacting personnel in a teaching veterinary hospital. J Equine Vet Sci 2024 134:105031.
    doi: 10.1016/j.jevs.2024.105031pubmed: 38336267google scholar: lookup
  27. Pereira V, Lopes C, Castro A, Silva J, Gibbs P, Teixeira P. Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal. Food Microbiol 2009 26:278–282.
    doi: 10.1016/j.fm.2008.12.008pubmed: 19269569google scholar: lookup
  28. SaraivaMMS, Rodrigues HLS, Benevides VP, de Leon CMCG, Santos SCL, Stipp DT, Givisiez PEN, Vieira RFC, Oliveira CJB. Antimicrobial resistance patterns of staphylococcus aureus cultured from the healthy horses’ nostrils sampled in distant regions of Brazil. Antibiotics 2025 14:693.
    doi: 10.3390/antibiotics14070693pubmed: 40723995pmc: 12291761google scholar: lookup
  29. Schilcher K, Horswill AR. Staphylococcal biofilm development: Structure, Regulation, and treatment strategies. Microbiol Mol Biol Rev 2020 12:e00026–e00019.
    doi: 10.1128/MMBR.00026-19google scholar: lookup
  30. Schwarz S, Feßler AT, Loncaric I, Wu C, Kadlec K, Wang Y, Shen J. Antimicrobial resistance among Staphylococci of animal origin. Microbiol Spectr 2018 6(4): ARBA-0010-2017.
  31. Shanson DC, Path FRC. Chap. 16 skin infections and infestations. Microbiol Clin Pract (Second Edition) 1989 374–399.
  32. Silveira DR, de Moraes TP, Kaefer K, Bach LG, de Oliveira Barbosa A, Moretti VD, de Menezes PQ, da Silva TT, Bandarra PM, Minello LF, Timm CD. MRSA and Enterobacteria of one health concern in wild animals undergoing rehabilitation. Res Soc Develop 2021 10:e34810111809.
    doi: 10.33448/rsd-v10i1.11809google scholar: lookup
  33. Štempelová L, Kubašová I, Bujňáková D, Kačírová J, Farbáková J, Maďar M, Karahutová L, Strompfová V. Distribution and characterization of Staphylococci isolated from healthy canine skin. Top Comp Anim Med 2022 49:100665.
  34. Stepanović S, Vuković D, Hola V, Bonaventura G, Djukić S, Ćirković I, Ruzicka F. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by Staphylococci. Apmis 2007 115:891–899.
  35. Strommenger B, Kettlitz C, Werner G, Witte W. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol 2003 41:4089–4094.
    doi: 10.1128/JCM.41.9.4089-4094.2003pubmed: 12958230pmc: 193808google scholar: lookup
  36. Strompfová V, Štempelová L. Composition and diversity of 16S rRNA basedskin bacterial microbiome in healthy horses. Vet Re Comm 2024 48:2847–2855.
  37. Strompfová V, Štempelová L, Bujňáková D, Karahutová L, Nagyová M, Siegfried L. Virulence determinants and antibiotic resistance in Staphylococci isolated from the skin of captive bred reptiles. Vet Res Comm 2024 48:1471–1480.
  38. Toledo-Arana A, Valle J, Solano C, Arrizubieta MJ, Cucarella C, Lamata M, Amorena B, Leiva J, Penadés JR, Lasa I. The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation. Appl Environ Microbiol 2001 67:4538–4545.
    doi: 10.1128/AEM.67.10.4538-4545.2001pubmed: 11571153pmc: 93200google scholar: lookup
  39. Yeh E, Pinsky BA, Banaei N, Baron EJ. Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests. PLoS ONE 2009 3:e6141.

Citations

This article has been cited 0 times.