biostudies-literatureUnknown14Aardal AM<h4>Introduction</h4>Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. <i>Providencia alcalifaciens</i> is suspected to play a role in the disease as it was commonly found in dogs suffering from AHDS during a Norwegian outbreak in 2019. The role of this bacterium as a constituent of the canine gut microbiota is unknown, hence this study set out to investigate its occurrence in healthy dogs using metagenomics.<h4>Materials and methods</h4>To decrease the likelihood of false detection, we established a metagenomic threshold for <i>P. alcalifaciens</i> by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for <i>P. alcalifaciens</i> was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for <i>P. alcalifaciens</i>. Finally, the metagenomic threshold was used to determine the occurrence of <i>P. alcalifaciens</i> in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project.<h4>Results</h4>The metagenomic assay and qPCR had a detection limit of 1.1x10³ CFU <i>P. alcalifaciens</i> per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique <i>k-</i>mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, <i>P. alcalifaciens</i> was found in only 1.1% (95% CI [0.0, 6.8]) of the samples, and then in low relative abundances (median: 0.04%; range: 0.00 to 0.81%). The sensitivity of the qPCR and shotgun metagenomics assay was low, as only 40% of culture-positive samples were also positive by qPCR and metagenomics.<h4>Discussion</h4>Using our detection limit, the occurrence of <i>P. alcalifaciens</i> in faecal samples from healthy dogs was low. Given the low sensitivity of the metagenomic assay, these results do not rule out a significantly higher occurrence of this bacterium at a lower abundance.Frontiers in cellular and infection microbiology1305742https://www.ebi.ac.uk/biostudies/studies/S-EPMC10933104biostudies-literatureDefining a metagenomic threshold for detecting low abundances of <i>Providencia alcalifaciens</i> in canine faecal samples.PMC10933104Soltvedt EMNorstebo SFRodriguez-Campos SSkancke EAardal AMHaverkamp THALlarena AKfalseDefining a metagenomic threshold for detecting low abundances of <i>Providencia alcalifaciens</i> in canine faecal samples.<h4>Introduction</h4>Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. <i>Providencia alcalifaciens</i> is suspected to play a role in the disease as it was commonly found in dogs suffering from AHDS during a Norwegian outbreak in 2019. The role of this bacterium as a constituent of the canine gut microbiota is unknown, hence this study set out to investigate its occurrence in healthy dogs using metagenomics.<h4>Materials and methods</h4>To decrease the likelihood of false detection, we established a metagenomic threshold for <i>P. alcalifaciens</i> by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for <i>P. alcalifaciens</i> was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for <i>P. alcalifaciens</i>. Finally, the metagenomic threshold was used to determine the occurrence of <i>P. alcalifaciens</i> in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project.<h4>Results</h4>The metagenomic assay and qPCR had a detection limit of 1.1x10³ CFU <i>P. alcalifaciens</i> per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique <i>k-</i>mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, <i>P. alcalifaciens</i> was found in only 1.1% (95% CI [0.0, 6.8]) of the samples, and then in low relative abundances (median: 0.04%; range: 0.00 to 0.81%). The sensitivity of the qPCR and shotgun metagenomics assay was low, as only 40% of culture-positive samples were also positive by qPCR and metagenomics.<h4>Discussion</h4>Using our detection limit, the occurrence of <i>P. alcalifaciens</i> in faecal samples from healthy dogs was low. Given the low sensitivity of the metagenomic assay, these results do not rule out a significantly higher occurrence of this bacterium at a lower abundance.2024-01-01T00:00:00Z20242025-04-04T19:16:17.88Z2025-02-19T04:40:40.988ZS-EPMC109331043848166310.3389/fcimb.2024.1305742