Development of a Species-Specific PCR Assay for Aerococcus urinaeequi Using Whole Genome Sequencing

Aerococcus urinaeequi is an opportunistic pathogen that has been isolated from humans, pigs, and chickens, but with no reports in geese until now. This research aimed to isolate and identify A. urinaeequi from four geese, and establish a specific PCR detection method for A. urinaeequi. Strain E1 was identified as A. urnaeequi through a combination of Gram staining (Gram-positive coccus), colony morphology (α-hemolysis), and whole genome sequencing analysis. Comparative genomics was used to analyze the genome sequences of five reference strains of A. urinaeequi to screen for a species-specific genomic region (401 bp). Based on this region, specific primers were designed to establish the PCR detection method for A. urnaeequi, and the specificity and sensitivity of this assay were tested. The results showed that the target sequence was specifically amplified only for the genome of A. urinaeequi, and that the minimum nucleic acid detection concentration was 7.08 × 10^-3 ng/μL. The mouse infection model indicated that the target fragment could be amplified from the tissue samples of dead mice in the challenge groups, verifying the applicability of PCR for clinical sample detection. Specific sequences of A. urinaeequi were detected in the lungs of three pigs using the PCR method, confirmed to be consistent through whole genome sequencing, and previously identified as A. urinaeequi or A. viridans by 16S rRNA sequencing. For the detection of fecal samples from geese, canines, and felines using the PCR method, the highest positive rate was 36.9% (31/84) of geese, followed by 21.7% (20/90) of felines, and finally 6.9% (16/230) of canines. A strain of A. urinaeequi was isolated and identified in geese for the first time, and a species-specific PCR detection method for A. urinaeequi was established with high specificity and sensitivity, which could well distinguish the bacterial species A. urinaeequi from its phylogenetically related species, A. viridans.