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Veterinary ophthalmology2020; 23(3); 480-488; doi: 10.1111/vop.12743

Characterization of the normal equine conjunctival bacterial community using culture-independent methods.

Abstract: The equine conjunctival microbiota has often been reported to be dominated by Gram-positive species such as Staphylococcus sp., Bacillus sp., and Corynebacterium sp. However, traditional culture-based methods can only recover a fraction of the bacterial species present in the sample. Objective: This pilot study aimed at exploring the diversity of the equine conjunctival microbiota using culture-independent methods. Methods: Eight horses were included in this study, and only eyes with normal ophthalmic examination (n = 15 eyes) were sampled. Methods: Conjunctival biopsies (culture-independent) were collected, and DNA was extracted from the tissues. Bacterial communities in conjunctival biopsies were characterized by next-generation sequencing of the 16S rRNA genes. Individual reads were ascribed to operational taxonomic units (OTUs) using BLASTn and Greengenes databases. Species richness, evenness, and Good's coverage were determined for each conjunctiva-associated microbial community. Results: Culture-independent samples produced a total of 329 bacterial OTUs. The main OTUs identified in the study belonged to the Gram-negative species Ralstonia mannitolilytica (88.0%), Nicoletella semolina (3.3%), and Pseudomonas tolaasii (1.5%). Conclusions: Contrary to previously published data based on culture-dependent methods, the horse eye microbial community was dominated by Gram-negative bacteria of the phylum Proteobacteria.
© 2020 American College of Veterinary Ophthalmologists.
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Publication Date: 2020-02-03 PubMed ID: 32017364DOI: 10.1111/vop.12743Google Scholar: Lookup
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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.

This study evaluates the diversity of bacteria found in the eyes of healthy horses, using culture-independent methods. It uncovers that the horse eye microbiota is predominantly composed of Gram-negative bacteria, which contradicts prior research based on culture-dependent methods that identified a dominance of Gram-positive bacteria.

Objective and Methods

The study sets out to understand more about the equine conjunctival microbiota, the party of bacteria present on the surface of horse eyes. Traditionally, this has been studied using culture-based methods which typically only highlight a portion of the bacteria. This study uses culture-independent methods, which aim to expose a broader range of bacteria. This involves testing the eyes of eight healthy horses (a total of 15 eyes). The conjunctiva of each horse was biopsied, the DNA extracted, and then characterized using next-generation sequencing of the 16S ribosomal RNA genes.

  • Conjunctival biopsies: literal tissue samples from the conjunctiva (the clear membrane covering the front of the eye).
  • 16S rRNA genes: these genes are a component in the production of ribosomes (the protein factories of the cell) and are found in all bacteria, which makes them ideal for identifying and comparing bacterial species.
  • Next-generation sequencing: a high-throughput method used to rapidly sequence large amounts of DNA or RNA.
  • BLASTn and Greengenes databases: public databases of genetic information used for comparison with sequenced samples.

Results and Findings

Unlike traditional culture-based methods, this culture-independent study discovered 329 bacterial operational taxonomic units (OTUs), a term referring to a type of bacteria. The main bacterial species identified were Gram-negative: Ralstonia mannitolilytica (88.0%), Nicoletella semolina (3.3%), and Pseudomonas tolaasii (1.5%). Gram-positive species, previously reported in other studies, were less prevalent.

  • Oxygen-dependent (hence aerobic) bacteria were more common than anaerobic (those species able to survive without strong quantities of oxygen) bacteria.
  • The biggest shifts in bacterial make-up were seen between individual horses rather than variations in the two eyes of the same horse.

Conclusion

The conclusions drawn from this study challenge previous understandings, suggesting that the microbial community within a horse’s eye is dominated by Gram-negative bacteria of the phylum Proteobacteria. The study highlights the benefit of using culture-independent methods, capable of revealing a more diverse bacterial profile.

Cite This Article

APA
LaFrentz S, Abarca E, Mohammed HH, Cuming R, Arias CR. (2020). Characterization of the normal equine conjunctival bacterial community using culture-independent methods. Vet Ophthalmol, 23(3), 480-488. https://doi.org/10.1111/vop.12743

Publication

Veterinary ophthalmology
ISSN: 1463-5224
NlmUniqueID: 100887377
Country: England
Language: English
Volume: 23
Issue: 3
Pages: 480-488

Researcher Affiliations

LaFrentz, Stacey
  • School of Fisheries, Aquaculture and Aquatic Sciences, Aquatic Microbiology Laboratory, Auburn University, Auburn, AL, USA.
Abarca, Eva
  • Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL, USA.
Mohammed, Haitham H
  • School of Fisheries, Aquaculture and Aquatic Sciences, Aquatic Microbiology Laboratory, Auburn University, Auburn, AL, USA.
Cuming, Rosemary
  • School of Fisheries, Aquaculture and Aquatic Sciences, Aquatic Microbiology Laboratory, Auburn University, Auburn, AL, USA.
Arias, Covadonga R
  • School of Fisheries, Aquaculture and Aquatic Sciences, Aquatic Microbiology Laboratory, Auburn University, Auburn, AL, USA.

MeSH Terms

  • Animals
  • Conjunctiva / microbiology
  • Female
  • Gram-Positive Bacteria / isolation & purification
  • Horses / microbiology
  • Male
  • Pilot Projects
  • Reference Values

Grant Funding

  • Alabama Agricultural Experiment Station
  • the School of Fisheries, Aquaculture, and Aquatic Sciences
  • the College of Veterinary Medicine at Auburn University

References

This article includes 71 references
  1. Gemensky-Metzler AJ, Wilkie DA, Kowalski JJ, Schmall LM, Willis AM, Yamagata M. Changes in bacterial and fungal ocular flora of clinically normal horses following experimental application of topical antimicrobial or antimicrobial-corticosteroid ophthalmic preparations.. Am J Vet Res 2005;66:800-811.
  2. Marchesi JR, Ravel J. The vocabulary of microbiome research: a proposal.. Microbiome 2015;3:31-31.
  3. Whitley RD, Moore CP. Microbiology of the equine eye in health and disease.. Vet Clin North Am Large Anim Pract 1984;6:451-466.
  4. Johns IC, Baxter K, Booler H, Hicks C, Menzies-Gow N. Conjunctival bacterial and fungal flora in healthy horses in the UK.. Vet Ophthalmol 2011;14:195-199.
  5. Moore CP, Heller N, Majors LJ, Whitley RD, Burgess EC, Weber J. Prevalence of ocular microorganisms in hospitalized and stabled horses.. Am J Vet Res 1988;49:773-777.
  6. Andrew SE, Nguyen A, Jones GL, Brooks DE. Seasonal effects on the aerobic bacterial and fungal conjunctival flora of normal thoroughbred brood mares in Florida.. Vet Ophthalmol 2003;6:45-50.
  7. McDonald PJ, Watson DJ. Microbial flora of normal canine conjunctivae.. J Small Anim Pract 1976;17:809-812.
  8. Espinola MB, Lilenbaum W. Prevalence of bacteria in the conjunctival sac and on the eyelid margin of clinically normal cats.. J Small Anim Pract 1996;37:364-366.
  9. Prado MR, Rocha MF, Brito EH. Survey of bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Fortaleza, Ceara, Brazil.. Vet Ophthalmol 2005;8:33-37.
  10. Urban M, Wyman M, Rheins M, Marraro RV. Conjunctival flora of clinically normal dogs.. J Am Vet Med Assoc 1972;161:201-206.
  11. Shewen PE, Povey RC, Wilson MR. A survey of the conjunctival flora of clinically normal cats and cats with conjunctivitis.. Can Vet J 1980;21:231-233.
  12. Spradbrow PB. The bacterial flora of the ovine conjunctival sac.. Aust Vet J 1968;44:117-118.
  13. Wilcox GE. Bacterial flora of the bovine eye with special reference to the Moraxella and Neisseria.. Aust Vet J 1970;46:253-256.
  14. Gouws JJ, Coetzer JA, Howell PG. A comparative microbiological study of clinically healthy eyes and those affected by ophthalmia in cattle and the association of noctuid eye-frequenting moths.. J S Afr Vet Assoc 1995;66:160-169.
  15. Davidson HJ, Rogers DP, Yeary TJ, Stone GG, Schoneweis DA, Chengappa MM. Conjunctival microbial flora of clinically normal pigs.. Am J Vet Res 1994;55:949-951.
  16. Cooper SC, McLellan GJ, Rycroft AN. Conjunctival flora observed in 70 healthy domestic rabbits (Oryctolagus cuniculus).. Vet Rec 2001;149:232-235.
  17. Coster ME, Stiles J, Krohne SG, Raskin RE. Results of diagnostic ophthalmic testing in healthy guinea pigs.. J Am Vet Med Assoc 2008;232:1825-1833.
  18. Davidson HJ, Vestweber JG, Brightman AH, Van Slyke TH, Cox LK, Chengappa MM. Ophthalmic examination and conjunctival bacteriologic culture results from a herd of North American bison.. J Am Vet Med Assoc 1999;215:1142-1144.
  19. Storms G, Meersschaert C, Farnir F, Grauwels M. Normal bacterial conjunctival flora in the Huacaya alpaca (Vicugna pacos).. Vet Ophthalmol 2015;19:22-28.
  20. Gionfriddo JR, Rosenbusch R, Kinyon JM, Betts DM, Smith TM. Bacterial and mycoplasmal flora of the healthy camelid conjunctival sac.. Am J Vet Res 1991;52:1061-1064.
  21. Montiani-Ferreira F, Mattos BC, Russ HH. Reference values for selected ophthalmic diagnostic tests of the ferret (Mustela putorius furo).. Vet Ophthalmol 2006;9:209-213.
  22. Montiani-Ferreira F, Truppel J, Tramontin MH, Vilani RG, Lange RR. The capybara eye: clinical tests, anatomic and biometric features.. Vet Ophthalmol 2008;11:386-394.
  23. Montiani-Ferreira F, Shaw G, Mattos BC, Russ HH, Vilani RG. Reference values for selected ophthalmic diagnostic tests of the capuchin monkey (Cebus apella).. Vet Ophthalmol 2008;11:197-201.
  24. Spinelli TP, Oliveira-Filho EF, Silva D, Mota R, Sa FB. Normal aerobic bacterial conjunctival flora in the Crab-eating raccoon (Procyon cancrivorus) and Coati (Nasua nasua) housed in captivity in pernambuco and paraiba (Northeast, Brazil).. Vet Ophthalmol 2010;13(Suppl):134-136.
  25. Tuntivanich P, Soontornvipart K, Tuntivanich N, Wongaumnuaykul S, Briksawan P. Conjunctival microflora in clinically normal Asian elephants in Thailand.. Vet Res Commun 2002;26:251-254.
  26. Leigue Dos Santos L, Montiani-Ferreira F, Lima L, Lange R, de Barros Filho IR. Bacterial microbiota of the ocular surface of captive and free-ranging microbats: Desmodus rotundus, Diameus youngi and Artibeus lituratus.. Vet Ophthalmol 2014;17:157-161.
  27. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation.. Microbiol Rev 1995;59:143-169.
  28. Willcox MD. Characterization of the normal microbiota of the ocular surface.. Exp Eye Res 2013;117:99-105.
  29. Graham JE, Moore JE, Jiru X. Ocular pathogen or commensal: a PCR-based study of surface bacterial flora in normal and dry eyes.. Invest Ophthalmol Vis Sci 2007;48:5616-5623.
  30. Dong Q, Brulc JM, Iovieno A. Diversity of bacteria at healthy human conjunctiva.. Invest Ophthalmol Vis Sci 2011;52:5408-5413.
  31. Zegans ME, Van Gelder RN. Considerations in understanding the ocular surface microbiome.. Am J Ophthalmol 2014;158:420-422.
  32. Thomason CA, Leon A, Kirkpatrick LT, Belden LK, Hawley DM. Eye of the Finch: characterization of the ocular microbiome of house finches in relation to mycoplasmal conjunctivitis.. Environ Microbiol 2017;19:1439-1449.
  33. Alfano N, Courtiol A, Vielgrader H, Timms P, Roca AL, Greenwood AD. Variation in koala microbiomes within and between individuals: effect of body region and captivity status.. Sci Rep 2015;5:10189.
  34. Weese SJ, Nichols J, Jalali M, Litster A. The oral and conjunctival microbiotas in cats with and without feline immunodeficiency virus infection.. Vet Res 2015;46:21-21.
  35. Leis ML, Costa MO. Initial description of the core ocular surface microbiome in dogs: Bacterial community diversity and composition in a defined canine population.. Vet Ophthalmol 2019;22:337-344.
  36. Scott EM, Arnold C, Dowell S, Suchodolski JS. Evaluation of the bacterial ocular surface microbiome in clinically normal horses before and after treatment with topical neomycin-polymyxin-bacitracin.. PLoS ONE 2019;14:e0214877-e0214877.
  37. Ploneczka-Janeczko K, Bania J, Bierowiec K, Kielbowicz M, Kielbowicz Z. Bacterial diversity in feline conjunctiva based on 16S rRNA gene sequence analysis: a pilot study.. BioMed Res Int 2017;2017:5.
  38. Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP).. 2016.
  39. Mackie RI, Wilkins CA. Enumeration of anaerobic bacterial microflora of the equine gastrointestinal-tract.. Appl Environ Microbiol 1988;54:2155-2160.
  40. Garner HE, Moore JN, Johnson JH. Changes in the caecal flora associated with the onset of laminitis.. Equine Vet J 1978;10:249-252.
  41. Ericsson AC, Johnson PJ, Lopes MA, Perry SC, Lanter HR. A Microbiological Map of the Healthy Equine Gastrointestinal Tract.. PLoS ONE 2016;11:e0166523.
  42. Costa MC, Silva G, Ramos RV. Characterization and comparison of the bacterial microbiota in different gastrointestinal tract compartments in horses.. Vet J 2015;205:74-80.
  43. Dougal K, de la Fuente G, Harris PA, Girdwood SE, Pinloche E, Newbold CJ. Identification of a core bacterial community within the large intestine of the horse.. PLoS ONE 2013;8(10):e77660.
  44. Boyer EEH. Studies on the bacterial flora of the mouth and nose of the normal horse.. J Bacteriol 1919;4:61-63.
  45. Baker GJ. A study of dental disease in the horse.. The University of Glasgow, Glasgow, Scotland, UK. [PhD Thesis]; 1979.
  46. Gao W, Chan Y, You M, Lacap-Bugler DC, Leung WK, Watt RM. In-depth snapshot of the equine subgingival microbiome.. Microb Pathog 2016;94:76-89.
  47. Kennedy R, Lappin DF, Dixon PM. The microbiome associated with equine periodontitis and oral health.. Vet Res 2016;47:49.
  48. Larsen AM, Bullard SA, Womble M, Arias CRJME. Community structure of skin microbiome of Gulf Killifish, Fundulus grandis, is driven by seasonality and not exposure to oiled sediments in a Louisiana Salt Marsh.. Microb Ecol 2015;70:534-544.
  49. Rosselló-Mora R, Amann R. The species concept for prokaryotes.. FEMS Microbiol Rev 2001;25:39-67.
  50. DeSantis TZ, Hugenholtz P, Larsen N. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.. Appl Environ Microbiol 2006;72:5069-5072.
  51. Schloss PD, Westcott SL, Ryabin T. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.. Appl Environ Microbiol 2009;75:7537-7541.
  52. Tamarzadeh A, Araghi-Sooreh A. Bacterial flora of the conjunctiva in healthy mules (Equus mulus).. Rev Med Vet 2014;165:334-337.
  53. Suter A, Voelter K, Hartnack S, Spiess BM, Pot SA. Septic keratitis in dogs, cats, and horses in Switzerland: associated bacteria and antibiotic susceptibility.. Vet Ophthalmol 2018;21:66-75.
  54. Whitley RD, Burgess EC, Moore CP. Microbial isolates of the normal equine eye.. Equine Vet J 1983;15:138-140.
  55. Laus F, Faillace V, Attili AR, Spaterna A, Tesei B, Cuteri V. Conjunctival bacterial and fungal flora in healthy donkeys in Central Italy.. Large Anim Rev 2016;22:137-142.
  56. Lu LJ, Liu J. Human microbiota and ophthalmic disease.. Yale J Biol Med 2016;89:325-330.
  57. Rodrigues Hoffmann A, Patterson AP, Diesel A. The skin microbiome in healthy and allergic dogs.. PLoS ONE 2014;9:e83197.
  58. Chiesa OA, Cuenca R, Mayayo E, Guarro J, Santamaria J, Stchigel AM. Cytological and microbiological findings in guttural pouch lavages of clinically normal horses with head restraint.. Aust Vet J 2002;80:234-238.
  59. Wada S, Hobo S, Ode H, Niwa H, Moriyama H. Equine keratomycosis in Japan.. Vet Ophthalmol 2013;16:1-9.
  60. Frontoso R, De Carlo E, Pasolini MP. Retrospective study of bacterial isolates and their antimicrobial susceptibilities in equine uteri during fertility problems.. Res Vet Sci 2008;84:1-6.
  61. Gomez GD, Balcazar JL. A review on the interactions between gut microbiota and innate immunity of fish.. FEMS Immunol Med Microbiol 2008;52:145-154.
  62. Mueller K, Ash C, Pennisi E, Smith O. The Gut Microbiota.. Science 2012;336:1245-1245.
  63. Llewellyn MS, Boutin S, Hoseinifar SH, Derome N. Teleost microbiomes: the state of the art in their characterization, manipulation and importance in aquaculture and fisheries.. Front Microbiol 2014;5:17.
  64. O'Neill AM, Gallo RL. Host-microbiome interactions and recent progress into understanding the biology of acne vulgaris.. Microbiome 2018;6:16.
  65. Zeeuwen P, Boekhorst J, van den Bogaard E. Microbiome dynamics of human epidermis following skin barrier disruption.. J Invest Dermatol 2012;132:S111-S111.
  66. Schommer NN, Gallo RL. Structure and function of the human skin microbiome.. Trends Microbiol 2013;21:660-668.
  67. Muro M, Soga Y, Higuchi T. Unusual oral mucosal microbiota after hematopoietic cell transplantation with glycopeptide antibiotics: potential association with pathophysiology of oral mucositis.. Folia Microbiol 2018;63:587-597.
  68. Klimesova K, Zakostelska ZJ, Tlaskalova-Hogenova H. Oral bacterial and fungal microbiome impacts colorectal carcinogenesis.. Front Microbiol 2018;9:13.
  69. Sultan AS, Kong EF, Rizk AM, Jabra-Rizk MA. The oral microbiome: A Lesson in coexistence.. PLoS Pathog 2018;14:6.
  70. Louca S, Doebeli M, Wegener Parfrey L. Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem.. Microbiome 2018;6:41.
  71. Větrovský T, Baldrian P. The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses.. PLoS ONE 2013;8:e57923-e57923.

Citations

This article has been cited 10 times.
  1. Thomson P, Pareja J, Núñez A, Santibáñez R, Castro R. Characterization of microbial communities and predicted metabolic pathways in the uterus of healthy mares. Open Vet J 2022 Nov-Dec;12(6):797-805.
    doi: 10.5455/OVJ.2022.v12.i6.3pubmed: 36650865google scholar: lookup
  2. Fernández-Garayzábal JF, LaFrentz S, Casamayor A, Abarca E, Mohammed HH, Cuming RS, Arias CR, Domínguez L, Vela AI. Corynebacterium conjunctivae: A New Corynebacterium Species Isolated from the Ocular Surface of Healthy Horses. Animals (Basel) 2022 Jul 18;12(14).
    doi: 10.3390/ani12141827pubmed: 35883374google scholar: lookup
  3. Santibáñez R, Lara F, Barros TM, Mardones E, Cuadra F, Thomson P. Ocular Microbiome in a Group of Clinically Healthy Horses. Animals (Basel) 2022 Apr 7;12(8).
    doi: 10.3390/ani12080943pubmed: 35454190google scholar: lookup
  4. Quiñones-Pérez C, Martínez A, Ortiz I, Crespo F, Vega-Pla JL. The Semen Microbiome and Semen Parameters in Healthy Stallions. Animals (Basel) 2022 Feb 22;12(5).
    doi: 10.3390/ani12050534pubmed: 35268102google scholar: lookup
  5. Quiñones-Pérez C, Hidalgo M, Ortiz I, Crespo F, Vega-Pla JL. Characterization of the seminal bacterial microbiome of healthy, fertile stallions using next-generation sequencing. Anim Reprod 2021;18(2):e20200052.
    doi: 10.1590/1984-3143-AR2020-0052pubmed: 34394753google scholar: lookup
  6. Martin de Bustamante MG, Plummer CE, Caddey B, Gomez DE. The effect of topical antibiotic or antibiotic-corticosteroid treatment on the ocular surface microbiota of healthy horses. Front Microbiol 2025;16:1535095.
    doi: 10.3389/fmicb.2025.1535095pubmed: 40831630google scholar: lookup
  7. Heun F, Meißner J, Schieder AK, Ohnesorge B, Busse C. Pantoea agglomerans in Equine Ulcerative Keratitis: Prevalence and Comparative Efficacy of Four Topical Antiseptics. Vet Ophthalmol 2026 Jan;29(1):e70044.
    doi: 10.1111/vop.70044pubmed: 40557447google scholar: lookup
  8. Płoneczka-Janeczko K, Armstrong E, Siemieniuch-Tartanus M, Magdziarz M. Remodelling of the healthy foal's conjunctival microbiome in the first two months of life. J Vet Res 2025 Mar;69(1):131-140.
    doi: 10.2478/jvetres-2025-0001pubmed: 40144056google scholar: lookup
  9. Farkas A, Thieme K, Soimala T, Jensen CK, Eule JC. Efficacy of 0.2% povidone-iodine and 0.1% polyhexamethylene biguanide as preoperative antiseptics in equine ophthalmic procedures. Vet Ophthalmol 2025 Mar;28(2):438-447.
    doi: 10.1111/vop.13306pubmed: 39648293google scholar: lookup
  10. Chow L, Flaherty E, Pezzanite L, Williams M, Dow S, Wotman K. Impact of Equine Ocular Surface Squamous Neoplasia on Interactions between Ocular Transcriptome and Microbiome. Vet Sci 2024 Apr 7;11(4).
    doi: 10.3390/vetsci11040167pubmed: 38668434google scholar: lookup
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