Full-length 16S ribosomal RNA sequencing of foal synovial fluid shows substantial agreement with microbial culture

Objective: To utilize full-length 16S rRNA long-read sequencing to (1) describe the microbiome profile of synovial fluid, previously classified by cytology and microbial culture, from foals with and without septic arthritis and (2) determine the agreement between the microbiome profiles and microbial culture. Methods: A retrospective observational study was conducted using synovial samples collected from foals treated at a single equine referral center (from 2018 through 2024). The synovial fluid was classified as septic or not septic using standard cytological and microbial culture methods. Deoxyribonucleic acid was extracted from stored samples and sequenced using a MinION sequencer. Linear mixed modeling was used to determine the relationships between α diversity and age, affected joint, and cytological and culture status. β diversity was assessed using permutational multivariate ANOVA. Differential taxa were identified with ALDEx2. The agreement between sequencing and culture was evaluated using the Gwet coefficient. Results: Fifty samples met inclusion criteria. α diversity (Shannon index) was higher in culture-positive samples (mean difference, 0.42; 95% CI, 0.12, 0.72). Microbiome composition differed by culture (R2 for Aitchison dissimilarity distance, 0.091) and cytology status (R2 for Jaccard dissimilarity distance, 0.029). No taxa differed by age, joint, or cytology. Klebsiella were significantly more abundant in culture-positive samples (ALDEx2 standardized effect size, 0.65; 95% CI, -1.63, 7.01). Agreement between positive culture and the predominant sequenced genus was 0.774 (Gwet AC1 coefficient) with a probabilistic benchmark interval of 0.6 to 0.8 (substantial agreement). Conclusions: Next-generation sequencing and culture results were substantially aligned. Clinical Relevance: Next-generation sequencing may aid diagnosis in suspected septic arthritis cases with negative culture results.