Determination of microbial diversity in the ocular conjunctiva of healthy dogs by total DNA sequencing

Marcela Torikachvili; Mariana Soares Silva; Michelle Becker Petersen; Fabiana Quoos Mayer; Renata da Fontoura Budaszewski; Matheus Nunes Weber; João Antonio Tadeu Pigatto; Cláudio Wageck Canal; Franciele Maboni Siqueira

doi:10.1590/1809-6891v25e-77549E

Autores

Marcela Torikachvili Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0002-5878-6857
Mariana Soares Silva Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0003-1830-9017
Michelle Becker Petersen Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0003-4857-5631
Fabiana Quoos Mayer Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0002-9324-8536
Renata da Fontoura Budaszewski Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0002-7102-358X
Matheus Nunes Weber Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0001-8282-6778
João Antonio Tadeu Pigatto Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0002-1541-9133
Cláudio Wageck Canal Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0002-0621-243X
Franciele Maboni Siqueira Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brasil https://orcid.org/0000-0001-7282-6117

DOI:

https://doi.org/10.1590/1809-6891v25e-77549E

Resumo

A conjuntiva desempenha papel fundamental na saúde e imunidade ocular e atua como uma barreira à entrada de microrganismos. As infecções conjuntivais são comuns em cães e resultam tanto da invasão de microrganismos patogênicos quanto do crescimento descontrolado da microbiota existente. A maior parte dos dados existentes provém de estudos baseados em métodos de cultura tradicionais. Esses relatos apontam para o predomínio de bactérias gram-positivas, principalmente Staphylococcus spp. O presente estudo analisou a comunidade microbiana presente na conjuntiva ocular de uma população heterogênea de cães sem distúrbios oftalmológicos por sequenciamento de DNA. Após exame oftálmico minucioso, foram coletados suabe conjuntivais de ambos os olhos de 30 cães. Após processamento e extração de ácidos nucleicos, o pool de amostras foi submetido ao sequenciamento shotgun de DNA por meio da plataforma Illumina e analisado no servidor Metagenomic Rapid Annotations using Subsystems Technology (MG-RAST). Foi identificada uma predominância do filo Proteobacteria e dos gêneros Ralstonia e Burkholderia juntamente com uma minoria de fungos, enquanto vírus não foram encontrados. O sequenciamento do DNA microbiano trouxe novos dados sobre o assunto, revelando a presença de organismos não cultiváveis até então desconhecidos como parte do microbioma ocular.

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Referências

Andrade AL, Stringhini G, Bonello FL, Marinho M, Perri SHV. Conjunctival microbiota of healthy dogs in Araçatuba city (SP) [Conjunctival microbiota of healthy dogs in Araçatuba city (SP)]. Arq Bras Oftalmol [Internet]. 2002 Jun [cited 2019 May 5];65:323–326. Available from: https://doi.org/10.1590/S0004-27492002000300008 Portuguese.

Galera PD, Ávila MO, Ribeiro CR, Santos FV.. Estudo da microbiota da conjuntiva ocular de macacos-prego (Cebus apella–LINNAEUS, 1758) e macacos bugio (Alouatta caraya–HUMBOLDT, 1812), provenientes do reservatório de Manso, MT, Brasil. Arq Inst Biol [Internet]. 2002 Apr [cited 2020 May 12];69:33-36. Available from: http://www.biologico.agricultura.sp.gov.br/uploads/docs/arq/V69_2/galera.pdf Portuguese.

Maggs DJ. Eyelids. In: Slatter’s Fundamentals of Veterinary Ophthalmology. 4. ed. St Louis: Saunders Elsevier; 2008. p. 107–134.

Armstrong RA. The microbiology of the eye. Ophthalmic Physiol Opt. 2000;6:429–441. https://doi.org/10.1111/j.1475-1313.2000.tb01121.x

Murphy JM, Lavach JD, Severin GA. Survey of conjunctival flora in dogs with clinical signs of external eye disease. J Am Vet Med Assoc. 1978;172:66-68. https://europepmc.org/article/med/624664

Ledbetter EC, Mun JJ, Kowbel D, Fleiszig SMJ.. Pathogenic phenotype and genotype of Pseudomonas aeruginosa isolates from spontaneous canine ocular infections. Invest Ophthalmol Vis Sci. 2009;50:729-736. https://doi.org/10.1167/iovs.08-2358

Oria AP, Pinna MH, Furtado MA, Pinheiro ACO, Gomes Junior DC, Costa Neto JM.. Microbiota conjuntival em cães clinicamente sadios e cães com ceratoconjuntivite seca. Ciênc Anim Bras. 2013;14:495-500. https://doi.org/10.5216/cab.v14I4.19210

Buyukmihci N. Ocular lesions of blastomycosis in the dog. J Am Vet Med Assoc [Internet]. 1982 Feb [cited 2021 Mar 19];180:426-431. Available from: https://escholarship.org/uc/item/8t04b5vh

Scott EM, Carter RT. Canine keratomycosis in 11 dogs: a case series (2000–2011). J Am Anim Hosp Assoc. 2014;50:112-118. https://doi.org/10.5326/JAAHA-MS-6012

Decaro N, Campolo M, Elia G, Buonavoglia D, Colaianni ML, Lorusso A, et al.Infectious canine hepatitis: an “old” disease reemerging in Italy. Res Vet Sci. 2007;83:269-273. https://doi.org/10.1016/j.rvsc.2006.11.009

Almeida DE, Roveratti C, Brito FL, Godoy GS, Duque JCM, Bechara GH, et al. Conjunctival effects of canine distemper virus‐induced keratoconjunctivitis sicca. Vet Ophthalmol. 2009;12:211-215 https://doi.org/10.1111/j.1463-5224.2009.00699.x

Ledbetter EC, Hornbuckle WE, Dubovi EJ. Virologic survey of dogs with naturally acquired idiopathic conjunctivitis. J Am Vet Med Assoc. 2009;235:954-959. https://doi.org/10.2460/javma.235.8.954

Ledbetter EC, Kim SG, Dubovi EJ, Bicalho RC. Experimental reactivation of latent canine herpesvirus-1 and induction of recurrent ocular disease in adult dogs. Vet Microbiol. 2009;138:98-105. https://doi.org/10.1016/j.vetmic.2009.03.013

McDonald PJ, Watson ADJ. Microbial flora of normal canine conjunctivae. J Small Anim Pract. 1976;17:809-812. https://doi.org/10.1111/j.1748-5827.1976.tb06947.x

Wang L, Pan Q, Zhang L, Xue Q, Cui J, Changming Q. Investigation of bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Beijing, China. Vet Ophthalmol. 2008;11:145-149. https://doi.org/10.1111/j.1463-5224.2008.00579.x

Santos LGF, Almeida ABPF, Silva MC, Oliveira JT, Dutra V, Souza VRF. Microbiota conjuntival de cães hígidos e com afecções oftálmicas [Conjunctival microorganisms from healthy dogs and with eye diseases]. Acta Sci Vet [Internet]. 2009 [cited 2022 May 18];37(2):165-169. Available from: https://www.redalyc.org/pdf/2890/289021830008.pdf

Verneuil M, Durand B, Marcon C, Guillot J. Conjunctival and cutaneous fungal flora in clinically normal dogs in southern France. J Mycol Med. 2014;24:25-28. https://doi.org/10.1016/j.mycmed.2013.11.002

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. https://doi.org/10.1111/vop.12599

Lima DA, Cibulski SP, Finkler F, Teixeira TF, Varela APM, Cerva C, et al. Faecal virome of healthy chickens reveals a large diversity of the eukaryote viral community, including novel circular ssDNA viruses. J Gen Virol. 2017;98:690-703. https://doi.org/10.1099/jgv.0.000711

Dong Q, Brulc JM, Iovieno A, Bates B, Garoutte A, Miller D, et al. Diversity of bacteria at healthy human conjunctiva. Invest Ophthal Vis Sci. 2011;52:5408-5413. https://doi.org/10.1167/iovs.10-6939

Huang Y, Yang B, Li W. Defining the normal core microbiome of conjunctival microbial communities. Clin Microbiol Infect. 2016;22:643-647. https://doi.org/10.1016/j.cmi.2016.04.008

Ozkan J, Nielsen S, Diez-Vives C, Coroneo M, Thomas T, Willcox M. Temporal stability and composition of the ocular surface microbiome. Sci Rep. 2017;7:1-11. https://doi.org/10.1038/s41598-017-10494-9

Vaz-Moreira I, Egas C, Nunes OC, Manaia CM. Culture-dependent and culture-independent diversity surveys target different bacteria: a case study in a freshwater sample. Antonie Van Leeuwenhoek. 2011;100:245-257. https://doi.org/10.1007/s10482-011-9583-0

Carter GR, Cole JR. Classification, normal flora and laboratory safety. In: Diagnostic procedures in veterinary bacteriology and mycology. 2. ed. San Diego: Academic Press; 1990. p. 1–8. https://doi.org/10.1016/B978-0-12-161775-2.50005-0

Kluge M, Campos FS, Tavares M, Amorim DB, Valdez FP, Giongo A, et al. Metagenomic survey of viral diversity obtained from faeces of Subantarctic and South American fur seals. PLoS One [Internet]. 2016 Mar [cited 2020 Aug 12];11:e0151921. Available from: https://doi.org/10.1371/journal.pone.0151921

Weber MN, Cibulski SP, Olegario JC et al. Characterization of dog serum virome from Northeastern Brazil. Virology. 2018;525:192-199. Available from: https://doi.org/10.1016/j.virol.2018.09.023

Alves CDBT, Budaszewski RF, Cibulski SP, Silva MS, Puhl DE, Mósena ACS, et al. Mamastrovirus 5 detected in a crab-eating fox (Cerdocyon thous): Expanding wildlife host range of astroviruses. Comp Immunol Microbiol Inf Dis. 2018;58:36-43. https://doi.org/10.1016/j.cimid.2018.08.002

Liu Z, Yang S, Wang Y, Shen Q, Yang Y, Deng X, et al. Identification of a novel human papillomavirus by metagenomic analysis of vaginal swab samples from pregnant women. Virol J. 2016;13:1-7. https://doi.org/10.1186/s12985-016-0583-6

Quinn PJ, Markey BK, Leonard FC et al. Nature, structure and taxonomy of viruses. In: Veterinary Microbiology and Microbial Disease. 2. ed. West Sussex: Wiley-Blackwell;2011. p. 505–508.

Suchodolski JS, Morris EK, Allenspach K, Jergens AE, Harmoinen JA, Westermarck E, et al. Prevalence and identification of fungal DNA in the small intestine of healthy dogs and dogs with chronic enteropathies. Vet Microbiol. 2008;132:379-388. https://doi.org/10.1016/j.vetmic.2008.05.017

Foster ML, Dowd SE, Stephenson C, Steiner JM, Suchodolski JS. Characterization of the fungal microbiome (mycobiome) in faecal samples from dogs. Vet Med Int [Internet]. 2013 Apr [cited 2023 July 09]:e658373:1-8. Available from: https://doi.org/10.1155/2013/658373

Prado MR, Rocha MF, Brito ÉH, Girão MD, Monteiro AJ, Teixeira MFS, et al. Survey of bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Fortaleza, Ceará, Brazil. Vet Ophthalmol. 2005;8:33-37. https://doi.org/10.1111/j.1463-5224.2005.04061.x

Allaker RP, Lloyd DH, Simpson AI. Occurrence of Staphylococcus intermedius on the hair and skin of normal dogs. Res Vet Sci. 1992;52:174-176. https://doi.org/10.1016/0034-5288(92)90006-N

Fazakerley J, Nuttall T, Sales D, Schmidt V, Carter SD, Hart CA, et al. Staphylococcal colonization of mucosal and lesional skin sites in atopic and healthy dogs. Vet Dermatol. 2009;20:179-184. https://doi.org/10.1111/j.1365-3164.2009.00745.x

Watanabe K, Nakaminami H, Azuma C, Tanaka I, Nakase K, Matsunaga N, et al. Methicillin-resistant Staphylococcus epidermidis is part of the skin flora on the hands of both healthy individuals and hospital workers. Biol Pharm Bull [Internet]. 2016 Aug [cited 2021 Apr 21]:b16-00528. Available from: https://doi.org/10.1248/bpb.b16-00528

Barrow GI. Microbial antagonism by Staphylococcus aureus. Microbiol. 1963;31:471-481. https://doi.org/10.1099/00221287-31-3-471

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;46:1-11. https://doi.org/10.1186/s13567-014-0140-5

Ryan MP, Adley CC. Ralstonia spp.: emerging global opportunistic pathogens. Eur J Clin Microbiol & Infect Dis. 2014;33:291-304. https://doi.org/10.1007/s10096-013-1975-9

Hoffmann AR, Patterson AP, Diesel A, Lawhon SD, Ly HJ, Stephenson CE, et al. The skin microbiome in healthy and allergic dogs. PloS one [Internet]. 2014 Jan [cited 2022 Jan 08]:9:e83197. Available from: https://doi.org/10.1371/journal.pone.0083197

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

Chen KJ, Sun MH, Hou CH, Sun CC, Chen TL. Burkholderia pseudomallei endophthalmitis. J Clin Microbiol. 2007;45:4073-4074. https://doi.org/10.1128/JCM.01467-07

Parkes HM, Shilton CM, Jerrett IV, Benedict S, Spratt BG, Godoy D, et al. Primary ocular melioidosis due to a single genotype of Burkholderia pseudomallei in two cats from Arnhem Land in the Northern Territory of Australia. J Feline Med Surg. 2009;11:856-863. https://doi.org/10.1016/j.jfms.2009.02.009

Lloyd JM, Suijdendorp P, Soutar WR. Melioidosis in a Dog. Aust Vet J. 1988;65:191-192. https://doi.org/10.1111/j.1751-0813.1988.tb14300.x

Makkar NS, Casida-Junior LE. Cupriavidus necator gen. nov., sp. nov.; a nonobligate bacterial predator of bacteria in soil. Int J Syst Evol Microbiol. 1987;37:323-326. https://doi.org/10.1099/00207713-37-4-323

Ray J, Waters RJ, Skerker JM, Kuehl JV, Price MN, Huang J, et al. Complete genome sequence of Cupriavidus basilensis 4G11, isolated from the Oak Ridge field research center site. mBio [Internet]. 2015 May [cited 2021 Oct 23]:3:e00322-15. Available from: https://doi.org/10.1128/genomeA.00322-15

Langevin S, Vincelette J, Bekal S, Gaudreau C. First case of invasive human infection caused by Cupriavidus metallidurans. J Clin Microbiol. 2011;49:744-745. https://doi.org/10.1128/JCM.01947-10

Andersson AM, Weiss N, Rainey F, Salkinoja-Salonen MS. Dust-borne bacteria in animal sheds, schools and children's day care centres. J Appl Microbiol. 1999;86:622-634. https://doi.org/10.1046/j.1365-2672.1999.00706.x

Cuscó A, Belanger JM, Gershony L, Islas-Trejo A, Levy K, Medrano JF, et al. Individual signatures and environmental factors shape skin microbiota in healthy dogs. Microbiome. 2017;5:1-15. https://doi.org/10.1186/s40168-017-0355-6

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 [Internet]. 2019 Apr [cited 2020 Mar 03]:14:e0214877. https://doi.org/10.1371/journal.pone.0214877