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

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.

Figure 1

Overview of samples included in the study, the methodology applied, and for which study aims the samples were used. In the sensitivity assay, faecal samples from dogs negative for Providencia alcalifaciens by cultivation were spiked with increasing amounts of P. alcalifaciens, from 1.1x10¹ to 1.1x10⁵ CFU, and thresholds for qPCR and metagenomics were established. The field control was used to test the established thresholds on faecal samples naturally contaminated with P. alcalifaciens, where samples from dogs positive and negative for P. alcalifaciens taken at different timepoints were compared using qPCR, metagenomics, and cultivation. Finally, the field study used the metagenomic abundance threshold to find the occurrence of P. alcalifaciens in a large population of dogs. Created with BioRender.com.

Figure 2

Bioinformatic pipeline used in this study, based on Talos (Haverkamp, 2020).

Figure 3

Box-and-whisker plots showing the distribution of reads and unique k-mer (Uk-mer) counts (A) and Uk-mers/reads ratios (B) for negative and spiked-in samples in the sensitivity assay. Uk-mer counts and Uk-mers/reads ratios were significantly different from negatives when 1.1x10³ CFU Providencia alcalifaciens or more were added to the samples (P < 0.05, marked by *).

Figure 4

(A) Scatterplot of Cq Providencia alcalifaciens from qPCR vs. CFU P. alcalifaciens for dog A and B, with regression showing a shared correlation coefficient R² of 0.64. (B) Scatterplot of genome equivalents P. alcalifaciens, as estimated from Cq values, vs. CFU P. alcalifaciens for dog A and B, with regressions showing correlation coefficients R² of 0.26 and 0.97, respectively.

Figure 5

(A) Scatterplot showing the distribution of reads, unique k-mers (Uk-mers) and Uk-mers/reads ratios vs. estimated CFU. Samples with estimated CFU > 3 logs for Providencia alcalifaciens had higher Uk-mer counts and Uk-mers/reads ratios than the samples with no CFU or low amounts of P. alcalifaciens. Linear regression used: Cq = 38.5−2.3xCFU ( Figure 4A ). (B) Box-and-whisker plots showing the distribution of reads and Uk-mers vs. cultivation status. (C) Scatterplot showing the relationship between Uk-mers/reads ratios vs. Uk-mers (log) by cultivation status. Samples with moderate and rich growth of P. alcalifaciens cluster together with high Uk-mers (log) and Uk-mers/reads ratios, separating clearly from samples with sparse and no growth.