EAR MICROBIOME OF DOGS WITH OTITIS: COMPARISON BETWEEN MICROBIOLOGICAL CULTURE AND BACTERIAL 16S RDNA SEQUENCING

Mário Tatsuo Makita; Theresse Camille Nascimento Holmström; Letícia Baptista Pinto; Gustavo Souza Lima Sant'Anna; Miriam Evelyn Castanheira De Faria; Luana de Oliveira Silva; Irene da Silva Coelho; Miliane Moreira Soares de Souza

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Trabalho

Título do Trabalho

EAR MICROBIOME OF DOGS WITH OTITIS: COMPARISON BETWEEN MICROBIOLOGICAL CULTURE AND BACTERIAL 16S RDNA SEQUENCING

Autores

Mário Tatsuo Makita
Theresse Camille Nascimento Holmström
Letícia Baptista Pinto
Gustavo Souza Lima Sant'Anna
Miriam Evelyn Castanheira De Faria
Luana de Oliveira Silva
Irene da Silva Coelho
Miliane Moreira Soares de Souza

Modalidade

Resumo

Área temática

Microbiologia e imunologia

Data de Publicação

10/03/2025

País da Publicação

Brasil

Idioma da Publicação

Inglês

Página do Trabalho

https://www.even3.com.br/anais/ix-simposio-internacional-em-ciencias-veterinarias-sincvet-ix-international-symposium-in-veterinary-sciences-isvs-499466/1033014-ear-microbiome-of-dogs-with-otitis--comparison-between-microbiological-culture-and-bacterial-16s-rdna-sequencing

ISBN

978-65-272-1246-1

Palavras-Chave

Canine otitis, treatment, antimicrobials

Resumo

Otitis is the most common ear disease in dogs. The condition can affect the outer, middle or inner ear and has multiple etiologies divided into primary, secondary, predisposing and perpetuating factors. Primary causes generally lead to imbalances, whether related to animal immunity, the microenvironment of the ear or the balance of the microbial community, and are responsible for initiating the inflammatory process, thus establishing otitis externa. The aim of this study was to analyze the ear microbiome of dogs affected by otitis externa using 16S rDNA sequencing and compare it with microbiological culture, to assess whether conventional culture is effective in identifying the main agent of otitis, and sufficient for an appropriate therapeutic approach. The samples were collected at private clinics, during routine clinical care, from animals with external otitis as their main or secondary complaint. The work was authorized by CEUA-UFRRJ under the number 9378290622. 40 samples were collected (18 from the right ear and 22 from the left ear) using a swab. Two swabs were taken from each ear, one for culture, stored in Stuart transport medium, and a second for molecular analysis, with the cotton tip cut off in a centrifuge microtube and kept frozen until arrival at the laboratory. The swabs intended for cultivation were inoculated onto 5% Sheep's Blood Agar and Mannitol Salt Agar and incubated at 37ºC for 24/48h. The bacterial isolates were identified using the MALDI-TOF method at the UFRJ Medical Microbiology Research Laboratory (LIMM). Total DNA was extracted manually using the phenol-chloroform technique adapted from Ayres, 2002. After extraction, the samples were evaluated using a spectrophotometer (Nano Drop ND-1000 Spectrophotometer Thermo Fisher Scientific) to check the quantity and quality of the DNA obtained and then stored frozen in a -20º C freezer. The integrity of the total DNA was assessed using 0.8% agarose gel electrophoresis. Library preparation of amplicons from the V3-V4 variable region of the 16S rDNA gene were generated by amplification with the primers Bakt_341F ('5-CCTAYGGGNGGCWGCAG-3') and Bakt_805R ('5-GACTACHVGGGTATCTAATCC-3') and sequencing on a 2x250 paired-end system on the NovaSeq platform (Illumina, USA), carried out at Novogene, Co, USA. The processed data was analyzed using Microbiome Analyst. Of the 40 samples collected, 18 had growth with only one bacterial species, 17 had mixed culture and in 5 samples there was no bacterial growth, resulting in 55 bacterial isolates. Comparing the results of the cultures with the sequencing, it was possible to see that 7.27% (4/55) bacterial isolates did not appear in the sequencing. Of the 55 isolates, 30.91% (17/55) were present in the culture and were compatible with the most abundant genus observed in the sequencing, and in 61.82% (34/55) isolates, the culture result was not compatible with the degree of abundance found. Among the non-compatible, negative and absent sequencing results, in 49.09% (27/43) cases there were cultivable genera that were more abundant in the sequencing than those isolated or not in the culture. For 29.09% (16/43) of the isolates, the most abundant bacteria were anaerobic. Since bacterial cultures of otitis are traditionally carried out under aerobic conditions, strict anaerobes end up not being isolated and are hardly related to cases of otitis. We can conclude that bacterial cultures of otitis are not always able to detect the most abundant bacterial population. In half of the non-compatible cases analyzed, the presence of more abundant culturable bacterial populations than those isolated was observed. We can therefore question the ability of microbiological cultures to detect the real infectious agent of otitis and whether these isolates are the best representatives for therapeutic analysis by antibiogram.

Título do Evento: IX Simpósio Internacional em Ciências Veterinárias (SINCVET) / IX International Symposium in Veterinary Sciences (ISVS)
Título dos Anais do Evento: Anais do IX Simpósio Internacional em Ciências Veterinárias (SINCVET) / IX International Symposium in Veterinary Sciences (ISVS)
Nome da Editora: Even3
Meio de Divulgação: Meio Digital

Como citar

MAKITA, Mário Tatsuo et al.. EAR MICROBIOME OF DOGS WITH OTITIS: COMPARISON BETWEEN MICROBIOLOGICAL CULTURE AND BACTERIAL 16S RDNA SEQUENCING.. In: Anais do IX Simpósio Internacional em Ciências Veterinárias (SINCVET) / IX International Symposium in Veterinary Sciences (ISVS). Anais...Seropédica(RJ) UFRRJ, 2025. Disponível em: https//www.even3.com.br/anais/ix-simposio-internacional-em-ciencias-veterinarias-sincvet-ix-international-symposium-in-veterinary-sciences-isvs-499466/1033014-EAR-MICROBIOME-OF-DOGS-WITH-OTITIS--COMPARISON-BETWEEN-MICROBIOLOGICAL-CULTURE-AND-BACTERIAL-16S-RDNA-SEQUENCING. Acesso em: 02/08/2025