Metagenomic and 16S rRNA analysis of culture-negative uterine samples identifies Brucella spp. in a female dog with pyometra

Canine pyometra is the most prevalent reproductive disease in female dogs and involves a complex interplay of hormonal and microbial factors. Although Enterobacteriaceae are known to be the main cause of pyometra, up to 25 % of samples remain negative when classic culture methods are used. The lack of growth in conventional media raises the intriguing possibility that non-cultivable microorganisms participate in the disease. Our study aimed to address this knowledge gap by investigating the microbiota in culture-negative uterine samples from dogs with pyometra. The purulent uterine contents of dogs with pyometra (n = 79) were subjected to bacterial culture. Most of the samples showed bacterial growth of Escherichia coli (48/79, 60.7 %) or other bacterial species (25/79, 31.8 %). Samples that tested negative using the classic culture method (6/79=7.5 %) were further analyzed using 16S rRNA gene sequencing, high-throughput 16S rRNA sequencing, and shotgun metagenomics. Only three of these provided DNA of sufficient quality for further analysis, which revealed a mixture of Haemophilus/Aggregatibacter and Fusobacterium/Porphyromonas in two uterine content. In the third sample, Brucella spp. was identified as the main genus. This study suggests that pathogens that are not easily isolated by classical culture methods, including zoonotic species, may be present in the uterus of dogs with pyometra.