Defining a metagenomic threshold for detecting low abundances of Providencia alcalifaciens in canine faecal samples

Front Cell Infect Microbiol. 2024 Feb 28:14:1305742. doi: 10.3389/fcimb.2024.1305742. eCollection 2024.

Abstract

Introduction: Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. Providencia alcalifaciens 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.

Materials and methods: To decrease the likelihood of false detection, we established a metagenomic threshold for P. alcalifaciens by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for P. alcalifaciens was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for P. alcalifaciens. Finally, the metagenomic threshold was used to determine the occurrence of P. alcalifaciens in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project.

Results: The metagenomic assay and qPCR had a detection limit of 1.1x103 CFU P. alcalifaciens per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique k-mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, P. alcalifaciens 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.

Discussion: Using our detection limit, the occurrence of P. alcalifaciens 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.

Keywords: AHDS; canine; clinical metagenomics; detection limit; faecal microbiota; shotgun sequencing.

MeSH terms

  • Animals
  • Bacteria / genetics
  • Diarrhea* / diagnosis
  • Diarrhea* / epidemiology
  • Diarrhea* / veterinary
  • Dogs
  • Feces / microbiology
  • Metagenome*
  • Metagenomics / methods
  • Providencia / genetics

Supplementary concepts

  • Providencia alcalifaciens

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The project was funded by NMBU Research Track Project funding (1354 020003), Astri og Birger Torsteds legat til fordel for dyrene and Agria research fund (N2020-0023). The analysis of the sequence data was performed on the HUNT Cloud computer cluster provided by the Norwegian University of Science and Technology. Funding for the HUNT One Health project was provided by the Ministry of Agriculture and Food, the Research Council of Norway, Nord-Trøndelag County authority, Norges Bondelag, Norsk Bonde- og Småbrukarlag, Animalia and the HUNT4-study.