Heterogeneity of gut microbial responses in healthy household dogs transitioning from an extruded to a mildly cooked diet

Introduction

For several millennia humans and dogs coevolved, sharing food and shelter (Axelsson et al., 2013). However, extruded dry food, commonly known as kibble, remains the most common form of feeding pet dogs in the United States. Extrusion cooking refers to a process where a mixture of ingredients is steam conditioned, compressed, and forced through the die of an extruder (Tran, Hendriks & Van der Poel, 2008). Mildly cooked (MC) diets, composed of ingredients that are cooked without extrusion, have been gaining in popularity in recent years among dog owners in the U.S. (PRWeb, 2015; Algya et al., 2018). Commercial MC canine diets are increasingly available, and must conform with the American Association Feed Control Officials (AAFCO) nutrient profiles or have been tested in feeding trials for the labeled life stages when complete and balanced. The nutritional profile of a MC diet may or may not differ significantly from dried and extruded pet food. Compared to dried and extruded food, MC diets have higher moisture content, higher digestibility of fats and protein, and dogs fed a MC diet for 28 days had lower levels of triglycerides (Algya et al., 2018; Oba et al., 2020; Do et al., 2021). Additionally, positive health perception was reported by dog owners after feeding their dogs a veterinarian-formulated MC diet (Johnson et al., 2016). However, the nutritional contents of MC diets and their effect on overall canine health remain understudied compared to other types of commercial dog foods.

Dietary changes are generally accompanied by alterations of the gut microbiota (GM) (Wernimont et al., 2020), which can have profound impact on canine health and disease (Redfern, Suchodolski & Jergens, 2017; Pilla & Suchodolski, 2019). Many studies have shown microbiome compositional changes upon switching to raw food diets, which are often higher in protein and fat and lower in carbohydrates (including fiber) than kibble diets (reviewed in (Wernimont et al., 2020)). These studies have shown that raw food diets lead to increased levels of certain Fusobacteria and Proteobacteria (Sandri et al., 2017; Algya et al., 2018; Schmidt et al., 2018). Similarly, MC diets have been associated with higher levels of certain Bacteroidetes, Fusobacteria and Proteobacteria, and lower levels of certain Actinobacteria and Firmicutes as compared to an extruded kibble diet (Algya et al., 2018; Do et al., 2021). However, these studies were conducted in only a small group of dogs under a laboratory setting. Thus, the extent that a MC diet influences the gut microbiomes of real world pet dogs with heterogeneous characteristics such as age, breeds, living conditions, and body size remains unexplored.

An investigation of the effect of dietary transition to a MC diet on GM and owner-reported subjective health metrics in healthy dogs was carried out to improve understanding of the effects of this increasingly popular diet type. Dogs switched from an extruded kibble to a MC diet for four weeks were assessed for changes relative to baseline in: (1) the gut microbiome by profiling the taxonomic and functional compositions of feces; and (2) perceived health outcomes using an owner-reported survey. Due to the variety of differences between diets, we hypothesized we would observe a shift in the diversity and abundance of GM upon diet change. Enrolled dogs were healthy at recruitment, however we aimed to determine if any additional positive impacts were reported by owners, including outcomes likely to change based on the literature for similar diets (e.g fecal output).

Materials and Methods

Dog owners gave electronic informed consent to the use of their dog’s non-identifying data for scientific study prior to study enrollment. All dog owners willingly underwent diet transition regardless of study participation. As previously explained (Tanprasertsuk et al., 2021), since fecal samples were collected non-invasively by the dog owners, no institutional animal ethical review was required.

Animals and study design

A total of 4,978 dog owners were contacted via email to gauge interest in study participation (Fig. 1). All owners were incoming customers of NomNomNow, Inc. (a direct-to-consumer pet food and health company in Nashville, TN, USA), who intended to switch their dog’s diet to a MC diet regardless of study participation. A total of 850 dogs of various breeds were screened using an online questionnaire; 54 dogs met the inclusion criteria described below and were enrolled in the study.

Dogs were eligible to participate in the study if they met the following owner-assessed criteria at enrollment: aged 1–14 years, body condition score (BCS) of 4–6 on the 9-point scale (Laflamme, 1997), currently feeding two meals per day exclusively of an extruded kibble diet with treats comprising ≤10% of overall caloric intake, absent of gastrointestinal (GI) issues (including chronic diarrhea/vomiting or diarrhea/vomiting within the previous 30 days), absent of any infections or major chronic diseases (pancreatitis, diabetes, kidney disease or failure, liver disease, heart disease, cancer, severe GI issues when young (<6 months old), and not pregnant or lactating). Only one dog per household was permitted to enroll. Dogs were not eligible to participate if they had consumed any prebiotics, probiotics, cultured foods, or antibiotics within the last three months, had a significant change in their diet within the last month, or had surgery within the previous three months. The criteria were similar to those previously described (Tanprasertsuk et al., 2021).

Participants were asked to provide the brand and formula of the baseline extruded kibble diet, and guaranteed analysis of nutrients was assembled when possible. Online health assessments were administered and stool samples were collected by owners one day before the diet transition (day 0) and again four weeks after the diet transition (day 28). On day 1, dogs began the transition from their previous extruded diet to a MC diet. An email reminder was sent out to all dog owners weekly to ensure adherence to the study diet and maintenance of baseline habits (supplements, medication, and exercise), as well as provide owners with the opportunity to report any observed adverse effects.

Results

Gut microbiome

Diversity

To investigate the effect of diet transition on GM α-diversity, we calculated two standard α-diversity measures, species richness and Shannon’s diversity index. A rarefaction curve generated for sequencing depth 10,000–470,000 reads demonstrated sufficient sequencing depth to assess the alpha diversity (Figs. S3A and S3B). Using sequencing depth at 470,000 reads, these α-diversity measures were not significantly different between baseline and four weeks after diet change (p > 0.05, Wilcoxon signed rank test).

PCoA was performed to investigate changes in β-diversity. The first two axes (PCo1 and PCo2), which respectively explained 34.2% and 10.3% of variation at the species level (Fig. 2A.) Eigenvalues for the first 25 PCo axes are displayed in Fig. S4. Spatial separation between the two timepoints along the first two PCo axes was demonstrated (p = 0.004, PERMANOVA Bray–Curtis dissimilarity matrices). A shift in PCo1 score, but not PCo2-5 scores, was also observed after 4 weeks of diet change (p = 1.72E−04, Wilcoxon signed rank test, Fig. 2B).

Using PAM Clustering analysis to investigate changes in β-diversity, the 56 samples (two samples from each of the 28 dogs) could be partitioned into two clusters (Fig. S5). The first cluster (C1) consisted primarily of baseline samples (15 samples collected at baseline and five samples collected at week 4), while the second cluster (C2) consisted of 13 samples collected at baseline and 23 samples collected at week 4. A significantly higher proportion of subjects had their baseline samples assigned to C1 and their week 4 samples assigned to C2 than the null hypothesis (p = 0.009, McNemar’s Chi-squared test). This result further confirms a significant shift in GM composition occurred due to dietary shift from extruded kibble to MC in these participants, although the presence of 13 baseline samples in C2 also indicates heterogeneous responses to diet change.

Relative abundances

Four phyla, Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes, dominated the GM (Fig. 3, 28 subjects are displayed in three separate rows based on the magnitude of MB response as explained in section Identification of high-,mid- and low-responders). No significant changes in relative abundances were observed between the timepoints at phylum or class levels after diet change (adjusted p > 0.05, negative binomial GLM with DESeq2). The relative abundance of the order Micrococcales significantly increased after the diet change (Table S3). At the family level, the relative abundances of four families significantly increased at week 4 (Leuconostocaceae, Hafniaceae, Lactobacillaceae, and Morganellaceae), while four families significantly decreased after 4 weeks of diet change (Prevotellaceae, Sutterellaceae, Atopobiaceae and Microbacteriaceae).

The effect of diet change was more pronounced at the genus and species levels. Relative abundances of 16 genera increased and 12 genera decreased significantly after the diet change (Table S3). Of the genera that increased, nine (56%) belong to the Enterobacterales order (Hafnia, Raoultella, Morganella, Proteus, Edwardsiella, Erwinia, Lelliottia, Yersinia, Leclercia), and seven (44%) belong to the Lactobacillales order (Leuconostoc, Tetragenococcus, Lactobacillus, Weissella, Carnobacterium, Pediococcus, Lactococcus). Half of the 12 decreasing genera were in the Bacillales order (Rummeliibacillus, Brevibacillus, Aneurinibacillus, Thermobacillus, Lysinbacillus, Bacillus). The relative abundances of 70 bacterial species significantly increased after the diet change (Fig. 4, Table 4). Among these species, 47 (67%) belong to the Lactobacillales order (also known as lactic acid bacteria or LAB), and 17 (24%) belong to the Enterobacterales order. Meanwhile, the relative abundances of 28 species significantly decreased, 13 (46%) of which belong to the Clostridiales order, 5 (18%) of which belong to the Bacillales order, and 3 (11%) of which belong to the Bacteroidales order.

Table 4:

Species with differential abundances between baseline and week 4 (n = 28, log2|fold change| ≥ 2 and FDR-adjusted p < 0.05).

Phylum	Class	Order	Family	Genus	Species	Relative abundance (%) Median (IQR)		Week 4 vs Baseline
						Baseline	Week 4	Log 2 FC mean ± SE	Adjusted P−value1
Increased at week 4 (70 species)
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	algidus	0.00E+00 (0.00E+00 to 1.64E−04)	3.54E−03 (1.48E−04 to 1.50E−01)	12.53 ± 1.11	1.12E−26
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	gelidum	5.01E−05 (0.00E+00 to 2.71E−04)	8.20E−03 (3.38E−04 to 1.43E+00)	11.90 ± 1.17	9.78E−22
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	sakei	2.62E−05 (0.00E+00 to 3.44E−04)	3.00E−03 (5.95E−05 to 7.40E−02)	11.61 ± 1.16	3.24E−21
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	fuchuensis	0.00E+00 (0.00E+00 to 5.26E−05)	1.12E−04 (0.00E+00 to 4.20E−03)	9.56 ± 1.34	1.85E−10
Proteobacteria	Gammaproteobacteria	Enterobacterales	Morganellaceae	Proteus	no data	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.06E−05)	9.50 ± 2.25	5.37E−04
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	carnosum	0.00E+00 (0.00E+00 to 1.80E−05)	3.22E−05 (0.00E+00 to 2.10E−02)	8.46 ± 1.43	2.85E−07
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	curvatus	0.00E+00 (0.00E+00 to 2.06E−04)	8.89E−04 (5.95E−05 to 1.83E−02)	7.46 ± 1.21	5.66E−08
Proteobacteria	Gammaproteobacteria	Enterobacterales	Morganellaceae	Proteus	mirabilis	0.00E+00 (0.00E+00 to 5.66E−05)	7.21E−05 (0.00E+00 to 1.79E−03)	7.32 ± 1.29	8.23E−07
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	siliginis	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.18E−03)	7.24 ± 1.43	1.76E−05
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	no data	0.00E+00 (0.00E+00 to 1.11E−04)	1.52E−03 (0.00E+00 to 3.58E−02)	7.19 ± 1.23	3.52E−07
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	kimchii	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 2.26E−03)	7.12 ± 1.60	2.28E−04
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	sanfranciscensis	0.00E+00 (0.00E+00 to 9.13E−05)	1.94E−04 (0.00E+00 to 6.55E−03)	6.94 ± 1.46	6.48E−05
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	pseudomesenteroides	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 7.94E−04)	6.94 ± 1.17	2.64E−07
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	citreum	2.35E−05 (0.00E+00 to 2.98E−04)	7.62E−04 (2.16E−04 to 2.33E−02)	6.68 ± 0.97	1.04E−09
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	fallax	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 7.02E−04)	6.67 ± 1.50	2.38E−04
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Tetragenococcus	halophilus	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 8.40E−05)	6.56 ± 1.74	2.81E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	saniviri	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 7.28E−05)	6.30 ± 1.77	5.03E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	plantarum	4.21E−05 (0.00E+00 to 2.57E−04)	1.37E−04 (0.00E+00 to 1.90E−03)	6.23 ± 1.12	1.48E−06
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	paralimentarius	0.00E+00 (0.00E+00 to 4.38E−05)	1.61E−05 (0.00E+00 to 1.75E−04)	6.22 ± 1.91	1.28E−02
Actinobacteria	Actinobacteria	Micrococcales	Micrococcaceae	Arthrobacter	no data	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 2.25E−04)	6.22 ± 1.42	2.90E−04
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Weissella	hellenica	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 6.72E−04)	6.15 ± 1.29	6.48E−05
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Weissella	cibaria	0.00E+00 (0.00E+00 to 6.30E−05)	2.25E−04 (0.00E+00 to 2.10E−03)	6.01 ± 1.18	1.61E−05
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	coryniformis	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 3.87E−04)	5.91 ± 1.46	9.81E−04
Proteobacteria	Gammaproteobacteria	Enterobacterales	Morganellaceae	Morganella	morganii	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 6.20E−05)	5.81 ± 1.37	4.88E−04
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	capillatus	0.00E+00 (0.00E+00 to 6.65E−05)	1.29E−05 (0.00E+00 to 2.49E−03)	5.81 ± 1.95	2.80E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Kosakonia	sp S29	0.00E+00 (0.00E+00 to 0.00E+00)	2.83E−05 (0.00E+00 to 1.35E−04)	5.76 ± 1.14	1.76E−05
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	mesenteroides	8.43E−05 (0.00E+00 to 2.37E−04)	3.02E−04 (0.00E+00 to 1.46E−02)	5.64 ± 1.07	5.87E−06
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	brevis	2.63E−05 (0.00E+00 to 1.26E−04)	9.84E−05 (0.00E+00 to 7.25E−04)	5.55 ± 0.97	7.77E−07
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	pentosus	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.39E−04)	5.43 ± 1.31	7.19E−04
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Weissella	jogaejeotgali	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.78E−04)	5.33 ± 1.43	3.02E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Pediococcus	inopinatus	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 9.05E−05)	5.08 ± 1.71	2.85E−02
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Weissella	confusa	0.00E+00 (0.00E+00 to 1.12E−04)	6.25E−05 (0.00E+00 to 1.90E−04)	4.92 ± 1.13	3.39E−04
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	oligofermentans	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 4.01E−04)	4.89 ± 1.50	1.25E−02
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Weissella	no data	0.00E+00 (0.00E+00 to 1.11E−04)	5.12E−05 (0.00E+00 to 6.32E−04)	4.86 ± 1.07	1.56E−04
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	no data	1.61E−03 (4.32E−04 to 4.28E−03)	1.57E−02 (7.43E−04 to 7.92E−02)	4.85 ± 0.72	1.94E−09
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	paraplantarum	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 2.48E−04)	4.80 ± 1.36	5.63E−03
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Weissella	paramesenteroides	0.00E+00 (0.00E+00 to 5.53E−05)	0.00E+00 (0.00E+00 to 2.43E−04)	4.70 ± 1.22	2.08E−03
Actinobacteria	Actinobacteria	Bifidobacteriales	Bifidobacteriaceae	Bifidobacterium	animalis	0.00E+00 (0.00E+00 to 1.59E−04)	0.00E+00 (0.00E+00 to 6.60E−06)	4.67 ± 1.68	4.39E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	hyovaginalis	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.18E−04)	4.57 ± 1.31	6.47E−03
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Weissella	koreensis	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 4.02E−04)	4.55 ± 1.59	3.43E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Hafnia	alvei	0.00E+00 (0.00E+00 to 7.61E−05)	9.66E−05 (0.00E+00 to 2.41E−03)	4.52 ± 1.42	1.58E−02
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	lactis	0.00E+00 (0.00E+00 to 2.35E−05)	7.59E−05 (0.00E+00 to 8.90E−04)	4.49 ± 1.26	5.03E−03
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	garlicum	0.00E+00 (0.00E+00 to 0.00E+00)	7.89E−05 (0.00E+00 to 1.36E−03)	4.45 ± 1.33	9.80E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Morganellaceae	Proteus	sp HMSC10D02	0.00E+00 (0.00E+00 to 6.23E−05)	0.00E+00 (0.00E+00 to 1.45E−04)	4.32 ± 1.10	1.65E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	parafarraginis	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 4.81E−05)	4.23 ± 1.40	2.59E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Raoultella	ornithinolytica	1.59E−05 (0.00E+00 to 3.26E−04)	1.35E−04 (0.00E+00 to 1.01E−03)	4.22 ± 0.94	2.04E−04
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	sp HMSC25G12	0.00E+00 (0.00E+00 to 9.44E−06)	0.00E+00 (0.00E+00 to 1.63E−04)	4.16 ± 1.38	2.59E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Edwardsiella	tarda	0.00E+00 (0.00E+00 to 4.38E−05)	7.19E−05 (0.00E+00 to 6.58E−04)	4.13 ± 1.20	7.62E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Cronobacter	dublinensis	0.00E+00 (0.00E+00 to 1.00E−04)	2.29E−05 (0.00E+00 to 2.66E−04)	4.07 ± 1.13	4.65E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp kppr-6	0.00E+00 (0.00E+00 to 7.30E−05)	3.30E−05 (0.00E+00 to 8.30E−05)	3.91 ± 1.16	9.79E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	kefiranofaciens	8.20E−05 (0.00E+00 to 2.80E−04)	1.43E−04 (2.66E−05 to 1.52E−03)	3.89 ± 1.37	3.57E−02
Firmicutes	Bacilli	Lactobacillales	Carnobacteriaceae	Carnobacterium	maltaromaticum	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.46E−04)	3.89 ± 0.92	5.42E−04
Firmicutes	Bacilli	Lactobacillales	Carnobacteriaceae	Carnobacterium	divergens	0.00E+00 (0.00E+00 to 1.58E−04)	1.63E−04 (0.00E+00 to 2.02E−03)	3.88 ± 1.04	2.99E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	cf. planticola B43	0.00E+00 (0.00E+00 to 3.96E−05)	2.68E−05 (0.00E+00 to 4.17E−05)	3.66 ± 1.01	4.25E−03
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	sp CL-2	3.68E−05 (0.00E+00 to 3.50E−04)	6.46E−05 (0.00E+00 to 6.24E−04)	3.62 ± 0.90	1.08E−03
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Lactococcus	piscium	1.70E−04 (2.10E−05 to 3.65E−04)	3.60E−04 (8.80E−05 to 4.30E−03)	3.32 ± 0.73	1.43E−04
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Raoultella	planticola	0.00E+00 (0.00E+00 to 7.77E−05)	6.66E−05 (0.00E+00 to 5.63E−04)	3.20 ± 1.00	1.48E−02
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	fermentum	4.90E−05 (0.00E+00 to 2.86E−04)	1.73E−04 (0.00E+00 to 5.50E−04)	3.16 ± 0.97	1.31E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Lelliottia	jeotgali	0.00E+00 (0.00E+00 to 3.33E−05)	3.11E−05 (0.00E+00 to 7.24E−05)	3.15 ± 1.13	4.39E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	cloacae complex Hoffmann cluster III	0.00E+00 (0.00E+00 to 2.78E−04)	1.62E−04 (0.00E+00 to 8.01E−04)	3.14 ± 0.96	1.25E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Tannerellaceae	Parabacteroides	merdae	1.54E−04 (0.00E+00 to 1.50E−03)	2.75E−05 (0.00E+00 to 1.05E−03)	3.12 ± 1.04	2.64E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Lactococcus	lactis	3.81E−03 (9.03E−04 to 2.35E−02)	7.09E−03 (2.76E−03 to 3.68E−02)	2.99 ± 0.84	5.03E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp HMSC067C01	1.45E−04 (0.00E+00 to 1.67E−03)	5.45E−04 (9.33E−05 to 1.77E−02)	2.93 ± 0.97	2.45E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	no data	1.44E−03 (0.00E+00 to 9.01E−03)	7.64E−03 (9.62E−04 to 2.58E−02)	2.92 ± 0.91	1.43E−02
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Pediococcus	acidilactici	2.62E−05 (0.00E+00 to 1.36E−04)	5.89E−05 (0.00E+00 to 4.94E−04)	2.79 ± 0.97	3.39E−02
Firmicutes	Clostridia	Clostridiales	Peptostreptococcaceae	Terrisporobacter	glycolicus	9.90E−05 (0.00E+00 to 1.49E−03)	2.07E−04 (0.00E+00 to 3.80E−03)	2.77 ± 0.97	3.47E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	asburiae	0.00E+00 (0.00E+00 to 5.62E−04)	2.42E−04 (3.63E−05 to 1.35E−03)	2.58 ± 0.89	3.18E−02
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	parabuchneri	8.76E−05 (0.00E+00 to 2.69E−04)	6.09E−05 (0.00E+00 to 2.71E−04)	2.36 ± 0.87	4.99E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Tyzzerella	nexilis	3.93E−02 (1.98E−02 to 5.81E−02)	2.04E−02 (4.61E−03 to 6.78E−02)	2.21 ± 0.61	4.25E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Pseudescherichia	vulneris	0.00E+00 (0.00E+00 to 8.89E−05)	1.10E−04 (3.63E−05 to 2.01E−04)	2.10 ± 0.77	4.86E−02
Decreased at week 4 (28 species)
Firmicutes	Bacilli	Bacillales	Planococcaceae	Rummeliibacillus	stabekisii	0.00E+00 (0.00E+00 to 8.97E−05)	0.00E+00 (0.00E+00 to 0.00E+00)	−10.85 ± 2.15	1.76E−05
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Paenibacillus	ginsengihumi	0.00E+00 (0.00E+00 to 1.49E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	−6.63 ± 1.37	4.55E−05
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Brevibacillus	borstelensis	0.00E+00 (0.00E+00 to 9.69E−05)	0.00E+00 (0.00E+00 to 0.00E+00)	−6.24 ± 1.54	9.81E−04
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Aneurinibacillus	no data	0.00E+00 (0.00E+00 to 1.84E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	−5.04 ± 1.42	5.03E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	canintestini	4.60E−05 (0.00E+00 to 3.30E−03)	1.06E−04 (0.00E+00 to 4.47E−04)	−5.02 ± 1.27	1.41E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	hirae	2.01E−04 (2.70E−05 to 5.32E−03)	1.40E−03 (0.00E+00 to 8.16E−03)	−4.48 ± 1.06	5.07E−04
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	copri	5.71E−05 (0.00E+00 to 1.20E−01)	2.94E−05 (0.00E+00 to 3.01E−04)	−4.41 ± 1.54	3.44E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	stercorea	0.00E+00 (0.00E+00 to 6.00E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	−3.97 ± 1.35	2.90E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	buccalis	0.00E+00 (0.00E+00 to 2.28E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	−3.91 ± 1.42	4.63E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Blautia	wexlerae	4.99E−02 (1.54E−02 to 1.35E−01)	2.52E−02 (5.28E−03 to 3.90E−02)	−3.90 ± 0.62	4.02E−08
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Blautia	sp Marseille-P2398	1.90E−03 (7.51E−04 to 4.41E−03)	6.13E−04 (0.00E+00 to 1.33E−03)	−3.75 ± 0.81	1.15E−04
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Blautia	no data	2.12E−01 (9.30E−02 to 3.45E−01)	6.61E−02 (8.53E−03 to 2.10E−01)	−3.33 ± 0.62	3.55E−06
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	thetaiotaomicron	1.96E−04 (3.66E−05 to 9.82E−04)	0.00E+00 (0.00E+00 to 1.73E−04)	−3.14 ± 0.88	5.03E−03
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	baratii	1.05E−04 (0.00E+00 to 5.42E−04)	3.42E−05 (0.00E+00 to 4.11E−04)	−2.99 ± 1.01	2.90E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	coprophilus	3.78E−04 (4.00E−05 to 2.33E−03)	2.95E−05 (0.00E+00 to 1.87E−04)	−2.96 ± 0.97	2.28E−02
Actinobacteria	Actinobacteria	Bifidobacteriales	Bifidobacteriaceae	Bifidobacterium	longum	1.30E−04 (0.00E+00 to 1.04E−03)	7.44E−05 (0.00E+00 to 5.36E−04)	−2.93 ± 1.00	3.15E−02
Firmicutes	Clostridia	Clostridiales	Peptostreptococcaceae	Peptostreptococcus	russellii	1.60E−04 (0.00E+00 to 5.47E−04)	0.00E+00 (0.00E+00 to 4.79E−05)	−2.92 ± 0.98	2.80E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Traorella	massiliensis	8.35E−05 (0.00E+00 to 2.42E−04)	5.39E−05 (0.00E+00 to 1.39E−04)	−2.88 ± 0.94	2.28E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	sp L2-50	4.28E−05 (0.00E+00 to 4.24E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	−2.88 ± 0.79	4.10E−03
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Blautia	obeum	2.29E−02 (9.54E−03 to 5.48E−02)	7.10E−03 (1.23E−03 to 2.10E−02)	−2.86 ± 0.59	4.88E−05
Firmicutes	Clostridia	Clostridiales	Ruminococcaceae	no data	no data	1.04E−03 (1.82E−04 to 4.44E−03)	1.81E−04 (0.00E+00 to 7.61E−04)	−2.75 ± 0.65	5.07E−04
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Roseburia	hominis	2.63E−04 (2.39E−05 to 1.91E−03)	9.08E−05 (0.00E+00 to 3.07E−04)	−2.74 ± 0.74	3.60E−03
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Faecalitalea	sp Marseille-P3755	1.04E−04 (0.00E+00 to 2.68E−04)	0.00E+00 (0.00E+00 to 5.27E−05)	−2.63 ± 0.91	3.20E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	no data	bacterium 28-4	5.43E−05 (2.65E−05 to 1.67E−04)	0.00E+00 (0.00E+00 to 4.85E−05)	−2.42 ± 0.73	1.07E−02
Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	no data	4.89E−04 (1.37E−04 to 1.13E−03)	5.78E−05 (0.00E+00 to 2.96E−04)	−2.40 ± 0.73	1.25E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Faecalicatena	contorta	7.58E−04 (7.63E−05 to 1.82E−03)	9.69E−05 (0.00E+00 to 4.14E−04)	−2.24 ± 0.77	3.18E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	no data	Bacterium 1 4 56FAA	2.36E−02 (8.91E−03 to 7.52E−02)	7.29E−03 (1.38E−03 to 1.72E−02)	−2.17 ± 0.67	1.32E−02
Firmicutes	Clostridia	Clostridiales	Ruminococcaceae	Faecalibacterium	prausnitzii	9.43E−03 (3.19E−03 to 2.18E−02)	1.46E−03 (1.30E−04 to 8.07E−03)	−2.07 ± 0.59	6.19E−03

DOI: 10.7717/peerj.11648/table-4

Notes:

1 P values were adjusted with false discovery rate for multiple comparisons

FC, fold change; SE, standard error.

Gut microbiome functionality

A total of 4,617 KO terms from all samples were identified, and after filtering 4,216 remained for subsequent analyses. PCoA was performed to investigate changes between time points. Fig. 5A shows the first two principal coordinate axes (which respectively explain 67.1% and 11.3% of the variation) and the eigenvalues for the first 25 axes are shown in Fig. S6. Spatial separation between the two timepoints along the first two PCoA axes was demonstrated (p = 0.007, PERMANOVA using the Bray–Curtis dissimilarity matrices). A significant shift in the PCo1 score after 4 weeks of diet change was observed (p = 0.013, Wilcoxon signed rank test, Fig. 5B). As listed in Table 5, a significant increase in the abundances of 14 KO terms was observed using a negative binomial GLM, while the abundances of 11 KO terms significantly decreased. Pathway mapping demonstrated differential abundances of pathways including the biosynthesis of secondary metabolites (ko01110: K02203, K13548, K16011), amino sugar and nucleotide sugar metabolism (ko00520: K13019, K16011, K17716), O-antigen nucleotide sugar biosynthesis (ko00541: K13019, K16011, K17716), and microbial metabolism in diverse environments (ko01120: K00385, K02203, K16165).

Table 5:

Kyoto Encyclopedia of Genes and Genomes Orthology (KO) terms with differential abundances between baseline and week 4 (n = 28, |fold change| ≥ 2 and adjusted p < 0.05).

KO term	Description	Week 4 vs. Baseline
		Log 2 FC mean ± SE	Adjusted P−value1
Increased abundance at week 4
K16323	purine nucleoside transport protein	7.2 ± 1.24	1.80E−05
K13548	2-deoxy-scyllo-inosamine dehydrogenase [EC:1.1.1.329]	6.29 ± 1.39	0.005
K11628	mycocerosic acid synthase [EC:2.3.1.111]	5.99 ± 1.58	0.034
K20707	lysine racemase [EC:5.1.1.5]	5.91 ± 1.62	0.046
K16959	L-cystine transport system permease protein	4.95 ± 1.15	0.009
K00638	chloramphenicol O-acetyltransferase type B [EC:2.3.1.28]	4.45 ± 1.14	0.025
K16846	(2R)-sulfolactate sulfo-lyase subunit beta [EC:4.4.1.24]	3.97 ± 0.97	0.016
K03457	nucleobase:cation symporter-1, NCS1 family	3.32 ± 0.72	0.004
K12442	phthiocerol/phenolphthiocerol synthesis type-I polyketide synthase C [EC:2.3.1.292]	3.13 ± 0.85	0.045
K12953	cation-transporting P-type ATPase F [EC:7.2.2.-]	3.07 ± 0.79	0.025
K19279	aminoglycoside 9-adenylyltransferase [EC:2.7.7.-]	2.66 ± 0.63	0.009
K18104	ATP-binding cassette, subfamily B, bacterial AbcA/BmrA [EC:7.6.2.2]	2.65 ± 0.68	0.025
K19422	glycosyltransferase EpsD [EC:2.4.-.-]	2.32 ± 0.59	0.025
K15125	filamentous hemagglutinin	1.91 ± 0.53	0.048
Decreased abundance at week 4
K13925	plasmin and fibronectin-binding protein A	−8.43 ± 1.90	0.006
K02203	phosphoserine / homoserine phosphotransferase [EC:3.1.3.3 2.7.1.39]	−5.08 ± 1.03	0.001
K13532	two-component system, sporulation sensor kinase D [EC:2.7.13.3]	−4.17 ± 1.15	0.047
K09759	nondiscriminating aspartyl-tRNA synthetase [EC:6.1.1.23]	−4.06 ± 0.78	0.000
K00351	Na+−transporting NADH:ubiquinone oxidoreductase subunit F [EC:7.2.1.1]	−3.88 ± 0.95	0.016
K14136	decaprenyl-phosphate phosphoribosyltransferase [EC:2.4.2.45]	−3.33 ± 0.87	0.030
K16165	fumarylpyruvate hydrolase [EC:3.7.1.20]	−2.97 ± 0.79	0.036
K16011	mannose-1-phosphate guanylyltransferase / mannose-6-phosphate isomerase [EC:2.7.7.13 5.3.1.8]	−2.65 ± 0.63	0.011
K13019	UDP-GlcNAc3NAcA epimerase [EC:5.1.3.23]	−2.01 ± 0.54	0.040
K17716	UDP-glucose 4-epimerase [EC:5.1.3.2]	−1.72 ± 0.46	0.034
K00385	anaerobic sulfite reductase subunit C	−1.64 ± 0.38	0.008

DOI: 10.7717/peerj.11648/table-5

Notes:

1 P values were adjusted with false discovery rate for multiple comparisons.

Differential gut microbiome responses to diet transition

Identification of high-, mid- and low-responders

While a significant shift in PCo1 score (0.16 ± 0.20) was observed across all subjects over the four week period, there were interindividual differences in the magnitude of the shift along PCo1 (Fig. 2C). Given this wide range of responses, subjects were further divided into tertiles based on the magnitude of PCo1 score change to examine the differential response. Those with the highest magnitude of positive change were termed high-responders (HR, n = 9), those in the second tertile mid-responders (MR, n = 9), and those in the third tertile low-responders (LR, n = 10). PCo1 score was significantly different among HR, MR, and LR at baseline (p = 0.017, Kruskal–Wallis test), but not at week 4 (p = 0.082, Kruskal–Wallis test). PCo1 score at baseline in HR was significantly lower than that of MR (FDR-adjusted p = 0.035, Dunn’s post hoc test) and LR (FDR-adjusted p = 0.025, Dunn’s post hoc test).

To better understand the observed differences in the GM composition among the differential response groups (HR, MR, and LR), the abundances at the species level of the 28 samples collected at baseline were further analyzed. PCoA was performed and the first two axes (PCo1 and PCo2) explained 37.4% and 10.0% of the variation. Spatial separation among HR, MR, and LR along the first two PCo axes was demonstrated (p = 0.022, PERMANOVA Bray–Curtis dissimilarity matrices) (Fig. S7). PAM Clustering analysis revealed two clusters. GM clusters using Bray–Curtis dissimilarity matrices and a MDS ordination assigned 8 HR, 2 MR and 2 LR samples to one cluster (C1), while 94% of samples assigned to the other cluster (C2) contained samples from MR or LR (p = 0.004, Fisher’s exact test). In other words, a significantly higher proportion of HR samples were assigned to C1 and a significantly higher proportion of MR and LR samples were assigned to C2. We also ran a random forest classification model on the samples collected at baseline (70% of the samples used as a training data set and 30% used as a validation data set), which was able to differentiate between the two classes (HR vs. MR/LR) with 78% accuracy. Classification error for HR and MR/LR were 33% and 17% respectively; AUC = 0.94. These results collectively demonstrate that the magnitude of GM response to diet change along the PCo1 axis was dependent on the initial gut microbiota.

Gut microbiome relative abundance at baseline in high- and low-responders

The relative abundances at the phylum level are displayed for HR, MR, and LR (Fig. 3). Comparisons of species abundances demonstrate a distinctive pattern between HR and LR at baseline (Table 6, Fig. 6A). Species belonging to the order Bacteroidales were observed to have higher, and never lower, abundances in HR as compared to LR (Fig. S8A). On the contrary, species belonging to the Enterobacterales or the Lactobacillales order were observed to have lower, and never higher, abundances in HR as compared to LR, with a single exception of Streptococcus minor (Lactobacillales order).

Table 6:

Bacterial species with differential abundances (|fold change| ≥ 2 and FDR-adjusted p < 0.05) between high-responders (HR, n = 9) and low-responders (LR, n = 10) at baseline.

Phylum	Class	Order	Family	Genus	Species	Relative abundance, in % Median (IQR)		Baseline HR vs LR
						HR (n = 9)	LR (n = 10)	Log 2 FC mean ± SE	Adjusted P−value1
Higher in HR (40 species)
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	sp 4 3 47FAA	3.88E−04 (5.24E−05 to 1.19E−02)	0.00E+00 (0.00E+00 to 2.96E−05)	10.02 ± 1.89	6.31E−06
Firmicutes	Clostridia	Clostridiales	Ruminococcaceae	Ruminiclostridium	josui	8.20E−04 (5.24E−05 to 7.18E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	7.37 ± 1.84	1.40E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	bivia	5.01E−04 (0.00E+00 to 3.85E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	7.29 ± 2.01	3.97E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	copri	2.09E−01 (1.01E−04 to 2.07E+00)	5.71E−05 (6.58E−06 to 1.21E−02)	7.06 ± 1.92	3.48E−03
Firmicutes	Negativicutes	Selenomonadales	Selenomonadaceae	Megamonas	sp Calf98-2	3.92E−01 (1.17E−02 to 5.17E−01)	8.40E−04 (0.00E+00 to 4.30E−03)	7.01 ± 1.67	9.19E−04
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	sp 3 1 33FAA	9.99E−04 (4.56E−05 to 1.46E−03)	0.00E+00 (0.00E+00 to 2.10E−05)	6.52 ± 1.49	4.47E−04
Firmicutes	Negativicutes	Selenomonadales	Selenomonadaceae	Megamonas	no data	3.38E+00 (1.43E−01 to 4.20E+00)	7.07E−03 (0.00E+00 to 4.27E−02)	6.38 ± 1.82	5.45E−03
Firmicutes	Negativicutes	Selenomonadales	Selenomonadaceae	Megamonas	funiformis	5.96E−01 (3.11E−02 to 7.66E−01)	1.25E−03 (0.00E+00 to 7.41E−03)	6.25 ± 1.71	3.53E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	buccalis	2.28E−04 (0.00E+00 to 7.57E−03)	0.00E+00 (0.00E+00 to 3.66E−05)	6.14 ± 1.53	1.40E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	sp 9 1 42FAA	1.82E−04 (6.11E−05 to 2.11E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	5.68 ± 1.81	1.45E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	vulgatus	6.41E−03 (2.69E−03 to 5.92E−02)	1.68E−04 (5.80E−05 to 2.84E−03)	5.63 ± 1.56	4.19E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	dorei	1.22E−03 (1.37E−04 to 1.38E−02)	7.04E−05 (0.00E+00 to 3.40E−04)	5.60 ± 1.62	5.82E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	no data	2.01E−03 (2.28E−04 to 7.25E−02)	0.00E+00 (0.00E+00 to 1.08E−04)	5.35 ± 1.73	1.57E−02
Firmicutes	Negativicutes	Selenomonadales	Selenomonadaceae	Megamonas	hypermegale	4.10E−03 (1.14E−04 to 8.73E−03)	0.00E+00 (0.00E+00 to 7.09E−05)	5.32 ± 1.48	4.22E−03
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Brevibacillus	panacihumi	4.48E−05 (0.00E+00 to 7.34E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	5.16 ± 2.02	4.77E−02
Actinobacteria	Actinobacteria	Micrococcales	Cellulomonadaceae	Cellulomonas	carbonis	1.82E−04 (0.00E+00 to 1.47E−03)	0.00E+00 (0.00E+00 to 2.96E−05)	5.10 ± 1.75	2.34E−02
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Brevibacillus	thermoruber	0.00E+00 (0.00E+00 to 1.14E−03)	0.00E+00 (0.00E+00 to 2.10E−05)	5.10 ± 1.82	2.88E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	bryantii	4.56E−05 (0.00E+00 to 9.71E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	5.00 ± 1.98	4.99E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Mediterranea	massiliensis	6.29E−04 (5.04E−04 to 1.95E−03)	0.00E+00 (0.00E+00 to 3.52E−05)	4.98 ± 1.50	9.25E−03
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Brevibacillus	borstelensis	1.11E−04 (0.00E+00 to 4.04E−03)	0.00E+00 (0.00E+00 to 7.20E−05)	4.96 ± 1.74	2.66E−02
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Paenibacillus	ginsengihumi	1.11E−04 (0.00E+00 to 4.84E−03)	0.00E+00 (0.00E+00 to 7.47E−05)	4.84 ± 1.67	2.47E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	stercorea	8.42E−04 (9.12E−05 to 6.78E−03)	0.00E+00 (0.00E+00 to 3.52E−05)	4.78 ± 1.52	1.42E−02
Firmicutes	Negativicutes	Selenomonadales	Selenomonadaceae	Megamonas	rupellensis	1.22E−01 (7.40E−03 to 1.67E−01)	2.47E−04 (0.00E+00 to 1.11E−03)	4.61 ± 1.72	3.58E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Tannerellaceae	Tannerella	sp 6 1 58FAA CT1	9.12E−05 (0.00E+00 to 5.49E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	4.58 ± 1.75	3.98E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Paraprevotella	clara	1.66E−04 (4.21E−05 to 7.94E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	4.57 ± 1.45	1.42E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	sp Marseille-P3166	1.47E−03 (3.03E−04 to 3.30E−03)	0.00E+00 (0.00E+00 to 2.45E−05)	4.42 ± 1.45	1.74E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Blautia	wexlerae	1.05E−01 (2.46E−02 to 3.83E+00)	1.98E−02 (1.25E−02 to 3.64E−02)	4.35 ± 0.96	2.41E−04
Bacteroidetes	Bacteroidia	Bacteroidales	Odoribacteraceae	Odoribacter	laneus	8.07E−04 (0.00E+00 to 2.87E−03)	1.40E−05 (0.00E+00 to 4.98E−05)	4.31 ± 1.43	1.87E−02
Proteobacteria	Betaproteobacteria	Burkholderiales	Sutterellaceae	Sutterella	no data	1.14E−03 (0.00E+00 to 1.26E−03)	4.21E−05 (9.86E−06 to 1.87E−04)	4.27 ± 1.57	3.52E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	multisaccharivorax	1.26E−04 (0.00E+00 to 2.91E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	4.18 ± 1.56	3.58E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	minor	1.27E−02 (1.85E−03 to 3.57E−02)	5.68E−04 (7.98E−05 to 1.16E−03)	3.94 ± 1.27	1.52E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Solobacterium	moorei	2.33E−03 (5.01E−04 to 1.45E−02)	0.00E+00 (0.00E+00 to 5.80E−05)	3.85 ± 1.51	4.79E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	sp AT4	8.65E−02 (6.45E−03 to 1.48E−01)	4.29E−03 (1.64E−03 to 8.18E−03)	3.78 ± 1.02	3.29E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	coprocola	2.32E−02 (7.57E−03 to 1.06E−01)	5.86E−04 (1.76E−04 to 2.05E−03)	3.64 ± 1.36	3.58E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Blautia	no data	6.00E−01 (1.76E−01 to 8.44E+00)	1.27E−01 (4.23E−02 to 2.47E−01)	3.63 ± 0.88	9.84E−04
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Blautia	obeum	5.40E−02 (1.96E−02 to 5.43E−01)	1.08E−02 (3.61E−03 to 2.24E−02)	3.30 ± 0.97	7.18E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	mediterraneensis	1.14E−03 (3.03E−04 to 2.57E−03)	5.38E−05 (9.01E−06 to 1.01E−04)	2.96 ± 1.08	3.41E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Marvinbryantia	formatexigens	3.58E−03 (8.07E−04 to 3.87E−03)	2.23E−04 (5.25E−05 to 4.42E−04)	2.78 ± 0.98	2.68E−02
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	barnesiae	7.33E−04 (2.28E−04 to 1.31E−03)	2.80E−05 (0.00E+00 to 7.93E−05)	2.75 ± 1.02	3.58E−02
Firmicutes	Clostridia	Clostridiales	Ruminococcaceae	no data	no data	3.05E−03 (7.17E−04 to 1.23E−02)	4.25E−04 (7.09E−05 to 1.13E−03)	2.43 ± 0.86	2.75E−02
Lower in HR (97 species)
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Hafnia	alvei	0.00E+00 (0.00E+00 to 0.00E+00)	1.32E−05 (0.00E+00 to 3.62E−04)	−26.49 ± 2.97	9.00E−17
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	braakii	0.00E+00 (0.00E+00 to 0.00E+00)	2.13E−03 (0.00E+00 to 8.85E−03)	−26.22 ± 2.42	7.87E−25
Proteobacteria	Gammaproteobacteria	Enterobacterales	Yersiniaceae	Serratia	marcescens	0.00E+00 (0.00E+00 to 0.00E+00)	1.51E−04 (6.58E−06 to 4.23E−03)	−25.50 ± 2.22	9.94E−28
Proteobacteria	Gammaproteobacteria	Pasteurellales	Pasteurellaceae	Haemophilus	sputorum	0.00E+00 (0.00E+00 to 0.00E+00)	2.80E−05 (0.00E+00 to 1.38E−03)	−24.56 ± 2.97	1.96E−14
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	sp MGH99	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 9.63E−04)	−23.88 ± 2.97	1.05E−13
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Hafnia	no data	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.97E−05)	−23.75 ± 2.97	1.24E−13
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	portucalensis	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.13E−04)	−23.27 ± 2.97	3.96E−13
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	cloacae complex sp ECNIH7	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 0.00E+00)	−22.05 ± 2.97	8.72E−12
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Edwardsiella	piscicida	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 1.97E−05)	−21.90 ± 2.97	1.14E−11
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	Cloaca complex sp TREC1	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 4.54E−04)	−21.17 ± 2.97	6.51E−11
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	canintestini	0.00E+00 (0.00E+00 to 0.00E+00)	9.29E−03 (1.63E−05 to 2.42E−01)	−9.45 ± 2.53	3.12E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	hormaechei	0.00E+00 (0.00E+00 to 0.00E+00)	2.04E−04 (0.00E+00 to 3.08E−03)	−9.31 ± 2.14	5.01E−04
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	dispar	0.00E+00 (0.00E+00 to 0.00E+00)	8.72E−04 (2.15E−04 to 6.38E−03)	−9.06 ± 1.73	8.87E−06
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	thailandicus	0.00E+00 (0.00E+00 to 0.00E+00)	2.71E−04 (5.50E−05 to 2.74E−03)	−8.92 ± 1.75	1.61E−05
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp FDAARGOS 163	0.00E+00 (0.00E+00 to 0.00E+00)	1.32E−05 (0.00E+00 to 7.07E−04)	−8.85 ± 2.20	1.40E−03
Firmicutes	Bacilli	Bacillales	Planococcaceae	Rummeliibacillus	stabekisii	0.00E+00 (0.00E+00 to 5.24E−05)	0.00E+00 (0.00E+00 to 4.72E−04)	−8.73 ± 2.93	2.00E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	asburiae	0.00E+00 (0.00E+00 to 0.00E+00)	4.31E−04 (0.00E+00 to 1.06E−03)	−8.61 ± 2.13	1.40E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp 4E1 DIV0656	0.00E+00 (0.00E+00 to 0.00E+00)	1.80E−05 (0.00E+00 to 1.27E−04)	−8.48 ± 2.54	8.51E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	sakei	0.00E+00 (0.00E+00 to 6.11E−05)	9.38E−05 (0.00E+00 to 3.41E−03)	−8.45 ± 2.03	9.81E−04
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	cloacae complex Hoffmann cluster III	0.00E+00 (0.00E+00 to 0.00E+00)	1.15E−04 (9.01E−06 to 1.22E−03)	−8.30 ± 1.93	6.02E−04
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Cronobacter	sakazakii	0.00E+00 (0.00E+00 to 0.00E+00)	1.01E−03 (3.61E−05 to 3.11E−03)	−8.03 ± 2.17	3.34E−03
Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Acinetobacter	indicus	0.00E+00 (0.00E+00 to 0.00E+00)	5.33E−05 (0.00E+00 to 8.20E−04)	−7.91 ± 2.27	5.56E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	sp ODB01	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 0.00E+00)	−7.82 ± 2.97	3.96E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp HMSC058D07	0.00E+00 (0.00E+00 to 0.00E+00)	6.00E−05 (0.00E+00 to 1.67E−03)	−7.80 ± 2.18	4.39E−03
Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	epidermidis	0.00E+00 (0.00E+00 to 0.00E+00)	4.53E−04 (6.70E−05 to 2.18E−03)	−7.76 ± 1.94	1.40E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	faecalis	0.00E+00 (0.00E+00 to 2.24E−04)	7.52E−03 (8.40E−05 to 3.24E−01)	−7.57 ± 2.15	5.26E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	quasipneumoniae	0.00E+00 (0.00E+00 to 0.00E+00)	1.05E−04 (3.75E−05 to 9.22E−04)	−7.43 ± 1.99	3.12E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	sp KTE151	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 3.29E−04)	−7.27 ± 2.70	3.58E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	freundii complex s CFNIH3	0.00E+00 (0.00E+00 to 0.00E+00)	3.29E−05 (0.00E+00 to 6.20E−04)	−7.25 ± 2.48	2.32E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	sp CL-2	0.00E+00 (0.00E+00 to 1.01E−04)	2.67E−05 (0.00E+00 to 2.68E−04)	−7.16 ± 2.25	1.32E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	sp S-77	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 4.98E−04)	−7.14 ± 2.67	3.58E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Erwiniaceae	Pantoea	sesami	0.00E+00 (0.00E+00 to 0.00E+00)	8.43E−05 (0.00E+00 to 1.47E−03)	−7.05 ± 2.64	3.58E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Franconibacter	helveticus	0.00E+00 (0.00E+00 to 0.00E+00)	2.50E−04 (9.01E−06 to 4.04E−04)	−6.90 ± 1.99	5.78E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	freundii complex sp CFNIH2	0.00E+00 (0.00E+00 to 0.00E+00)	2.99E−04 (6.31E−05 to 5.02E−04)	−6.88 ± 1.94	4.73E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	werkmanii	0.00E+00 (0.00E+00 to 0.00E+00)	5.38E−05 (9.01E−06 to 3.96E−04)	−6.70 ± 1.82	3.43E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	cloacae complex Hoffmann cluster IV	0.00E+00 (0.00E+00 to 0.00E+00)	3.91E−04 (4.80E−05 to 8.27E−04)	−6.70 ± 1.83	3.56E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	curvatus	1.14E−04 (0.00E+00 to 1.79E−04)	1.64E−04 (0.00E+00 to 3.71E−03)	−6.69 ± 1.71	1.80E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	michiganensis	0.00E+00 (0.00E+00 to 0.00E+00)	6.77E−04 (1.45E−04 to 2.32E−03)	−6.60 ± 1.65	1.40E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Pluralibacter	gergoviae	0.00E+00 (0.00E+00 to 0.00E+00)	6.58E−05 (8.17E−06 to 5.18E−04)	−6.56 ± 1.75	3.12E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Shigella	no data	0.00E+00 (0.00E+00 to 0.00E+00)	1.67E−01 (1.76E−02 to 1.01E+00)	−6.46 ± 2.31	2.88E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Erwiniaceae	Erwinia	teleogrylli	0.00E+00 (0.00E+00 to 0.00E+00)	9.93E−05 (8.17E−06 to 6.65E−04)	−6.45 ± 1.71	3.12E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp KTE96	0.00E+00 (0.00E+00 to 0.00E+00)	4.75E−04 (9.01E−06 to 3.41E−03)	−6.43 ± 2.21	2.43E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp KTE11	0.00E+00 (0.00E+00 to 0.00E+00)	1.40E−05 (0.00E+00 to 2.35E−03)	−6.38 ± 2.38	3.58E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Shigella	sonnei	0.00E+00 (0.00E+00 to 0.00E+00)	9.59E−02 (9.56E−03 to 2.82E−01)	−6.38 ± 2.21	2.52E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp FDAARGOS 375	0.00E+00 (0.00E+00 to 0.00E+00)	6.27E−05 (0.00E+00 to 4.75E−04)	−6.31 ± 2.28	3.09E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Cronobacter	malonaticus	0.00E+00 (0.00E+00 to 0.00E+00)	6.41E−05 (1.05E−05 to 1.06E−03)	−6.27 ± 1.99	1.42E−02
Proteobacteria	Betaproteobacteria	Burkholderiales	Alcaligenaceae	Achromobacter	sp ATCC35328	0.00E+00 (0.00E+00 to 0.00E+00)	9.52E−03 (1.18E−03 to 1.99E−02)	−6.16 ± 2.04	1.89E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	oxytoca	0.00E+00 (0.00E+00 to 0.00E+00)	7.20E−04 (2.11E−05 to 3.25E−03)	−6.16 ± 1.88	1.04E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp KTE159	0.00E+00 (0.00E+00 to 0.00E+00)	1.01E−02 (2.82E−04 to 3.19E−02)	−6.16 ± 2.14	2.54E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	no data	0.00E+00 (0.00E+00 to 0.00E+00)	6.56E−03 (2.91E−04 to 3.21E−02)	−6.15 ± 2.21	3.05E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	massiliensis	0.00E+00 (0.00E+00 to 9.71E−05)	1.97E−04 (3.94E−05 to 8.56E−04)	−6.06 ± 1.79	7.55E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	no data	no data	2.28E−04 (9.71E−05 to 4.19E−04)	8.92E+00 (5.93E−01 to 3.02E+01)	−6.05 ± 1.81	8.53E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	hirae	8.42E−05 (0.00E+00 to 2.62E−04)	3.64E−04 (4.34E−05 to 4.62E−03)	−5.98 ± 1.67	4.37E−03
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	equinus	4.55E−04 (1.94E−04 to 1.50E−03)	1.76E−03 (1.06E−04 to 8.90E−01)	−5.94 ± 1.93	1.61E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	raffinosus	0.00E+00 (0.00E+00 to 0.00E+00)	8.85E−05 (0.00E+00 to 6.18E−04)	−5.90 ± 2.19	3.58E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp KTE52	0.00E+00 (0.00E+00 to 0.00E+00)	2.20E−03 (1.54E−04 to 1.79E−02)	−5.90 ± 1.97	1.94E−02
Actinobacteria	Actinobacteria	Propionibacteriales	Propionibacteriaceae	Propionibacterium	freudenreichii	1.11E−04 (0.00E+00 to 2.02E−04)	6.80E−05 (1.05E−05 to 2.17E−03)	−5.89 ± 1.84	1.29E−02
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Paenibacillus	sp VT-16-81	0.00E+00 (0.00E+00 to 0.00E+00)	3.73E−05 (0.00E+00 to 1.08E−04)	−5.87 ± 2.04	2.54E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Shigella	boydii	0.00E+00 (0.00E+00 to 0.00E+00)	8.74E−02 (7.11E−03 to 2.18E−01)	−5.87 ± 2.18	3.58E−02
Firmicutes	Bacilli	Lactobacillales	Carnobacteriaceae	Carnobacterium	gallinarum	0.00E+00 (0.00E+00 to 0.00E+00)	3.77E−05 (0.00E+00 to 7.99E−04)	−5.85 ± 2.17	3.58E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	lutetiensis	8.42E−05 (0.00E+00 to 4.62E−03)	1.49E−04 (6.23E−05 to 3.44E+00)	−5.85 ± 2.30	4.89E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp HMSC34G12	0.00E+00 (0.00E+00 to 0.00E+00)	7.47E−05 (0.00E+00 to 2.38E−04)	−5.83 ± 2.08	2.88E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	no data	3.68E−02 (8.00E−03 to 5.84E−01)	1.91E−01 (1.39E−02 to 2.36E+01)	−5.81 ± 1.55	3.12E−03
Fusobacteria	Fusobacteria	Fusobacteriales	Leptotrichiaceae	Streptobacillus	moniliformis	0.00E+00 (0.00E+00 to 0.00E+00)	3.73E−03 (6.84E−04 to 1.09E−02)	−5.78 ± 1.92	1.91E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp 1 1 43	0.00E+00 (0.00E+00 to 0.00E+00)	8.41E−03 (8.32E−04 to 9.78E−03)	−5.68 ± 1.83	1.52E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	infantarius	8.55E−04 (0.00E+00 to 1.39E−03)	6.33E−04 (1.34E−04 to 8.77E−01)	−5.65 ± 2.00	2.75E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	canis	0.00E+00 (0.00E+00 to 0.00E+00)	3.32E−04 (9.01E−06 to 1.50E−03)	−5.59 ± 2.16	4.44E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp HMSC035C10	0.00E+00 (0.00E+00 to 9.12E−05)	5.28E−04 (4.21E−05 to 9.66E−04)	−5.59 ± 1.77	1.42E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	villorum	0.00E+00 (0.00E+00 to 8.42E−05)	2.35E−05 (0.00E+00 to 1.47E−04)	−5.56 ± 2.06	3.58E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp 255 ESPC	0.00E+00 (0.00E+00 to 0.00E+00)	1.52E−04 (9.01E−06 to 4.48E−03)	−5.54 ± 1.92	2.47E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Lelliottia	amnigena	0.00E+00 (0.00E+00 to 0.00E+00)	9.27E−05 (1.89E−05 to 3.69E−04)	−5.53 ± 1.84	1.91E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	anginosus	0.00E+00 (0.00E+00 to 8.42E−05)	7.61E−04 (1.17E−05 to 3.15E−03)	−5.46 ± 1.77	1.58E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Erwiniaceae	Erwinia	gerundensis	0.00E+00 (0.00E+00 to 0.00E+00)	9.82E−05 (0.00E+00 to 1.26E−04)	−5.45 ± 1.82	1.92E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	no data	0.00E+00 (0.00E+00 to 0.00E+00)	3.09E−03 (3.87E−04 to 1.20E−02)	−5.39 ± 2.00	3.58E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp HMSC072F02	0.00E+00 (0.00E+00 to 5.05E−04)	5.25E−04 (8.41E−05 to 6.68E−03)	−5.29 ± 1.68	1.42E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	no data	bacterium ENNIH1	0.00E+00 (0.00E+00 to 0.00E+00)	5.49E−05 (0.00E+00 to 1.18E−04)	−5.28 ± 1.85	2.60E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Raoultella	planticola	0.00E+00 (0.00E+00 to 0.00E+00)	1.89E−05 (0.00E+00 to 1.16E−04)	−5.17 ± 2.03	4.79E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	gallinarum	0.00E+00 (0.00E+00 to 5.55E−05)	2.51E−03 (1.32E−03 to 1.13E−02)	−5.12 ± 1.57	1.04E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Yersiniaceae	Yersinia	no data	0.00E+00 (0.00E+00 to 0.00E+00)	1.41E−04 (9.01E−06 to 2.87E−04)	−5.08 ± 1.86	3.41E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Raoultella	ornithinolytica	0.00E+00 (0.00E+00 to 0.00E+00)	9.13E−05 (9.44E−06 to 7.79E−04)	−5.06 ± 1.90	3.62E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	no data	6.11E−05 (0.00E+00 to 2.10E−04)	1.21E+00 (5.58E−01 to 7.20E+00)	−5.02 ± 1.99	4.99E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	sp MGH 55	0.00E+00 (0.00E+00 to 0.00E+00)	5.88E−05 (9.01E−06 to 2.40E−04)	−5.00 ± 1.77	2.75E−02
Actinobacteria	Actinobacteria	Corynebacteriales	Nocardiaceae	Rhodococcus	no data	5.55E−05 (0.00E+00 to 3.14E−04)	2.03E−04 (0.00E+00 to 7.70E−04)	−4.89 ± 1.50	1.04E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	thermophilus	1.82E−04 (6.11E−05 to 6.81E−04)	1.08E−02 (5.86E−04 to 2.09E−02)	−4.67 ± 1.35	6.07E−03
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	macedonicus	1.01E−04 (0.00E+00 to 1.48E−02)	2.99E−03 (4.25E−04 to 6.15E−02)	−4.62 ± 1.70	3.52E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	pasteurianus	8.73E−04 (5.69E−04 to 3.33E−03)	6.32E−03 (4.42E−04 to 4.98E−02)	−4.60 ± 1.17	1.80E−03
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	suis	3.88E−04 (3.19E−04 to 9.27E−04)	8.34E−04 (2.27E−04 to 3.00E−03)	−4.10 ± 1.03	1.40E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	durans	1.26E−04 (0.00E+00 to 3.19E−04)	7.94E−04 (4.20E−05 to 2.29E−03)	−4.04 ± 1.53	3.96E−02
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	no data	5.92E−04 (2.53E−04 to 1.92E−03)	3.83E−03 (1.86E−03 to 1.66E−02)	−4.00 ± 1.07	3.12E−03
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Lactococcus	lactis	1.26E−03 (8.88E−04 to 3.54E−03)	2.40E−02 (3.15E−03 to 2.71E−02)	−3.96 ± 0.95	9.52E−04
Firmicutes	Clostridia	Clostridiales	Clostridiales	Emergencia	timonensis	3.05E−04 (1.37E−04 to 7.34E−04)	4.46E−04 (2.25E−04 to 1.29E−03)	−3.84 ± 1.16	9.41E−03
Actinobacteria	Actinobacteria	Bifidobacteriales	Bifidobacteriaceae	Bifidobacterium	breve	3.05E−03 (1.65E−03 to 3.41E−03)	1.63E−03 (6.32E−04 to 4.35E−03)	−3.79 ± 0.99	2.64E−03
Firmicutes	Bacilli	no data	no data	no data	no data	8.66E−04 (1.34E−04 to 4.72E−03)	4.48E−03 (1.65E−03 to 3.28E−02)	−3.73 ± 1.28	2.34E−02
Proteobacteria	Gammaproteobacteria	no data	no data	no data	no data	5.24E−05 (4.21E−05 to 1.14E−04)	1.39E−02 (3.37E−03 to 3.15E−02)	−3.73 ± 1.29	2.47E−02
Actinobacteria	Actinobacteria	Micrococcales	Microbacteriaceae	Microbacterium	no data	5.55E−05 (0.00E+00 to 2.91E−04)	9.61E−05 (3.85E−05 to 3.39E−04)	−2.94 ± 1.14	4.61E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	no data	bacterium 3 1 46FAA	1.14E−02 (2.69E−03 to 2.41E−02)	1.05E−02 (1.24E−03 to 6.32E−02)	−2.78 ± 0.99	2.75E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	parasanguinis	7.76E−04 (6.72E−04 to 1.21E−03)	8.33E−04 (3.19E−04 to 1.59E−03)	−2.19 ± 0.71	1.58E−02

DOI: 10.7717/peerj.11648/table-6

Notes:

1 P values were adjusted with false discovery rate for multiple comparisons.

FC, fold change; SE, standard error.

Bacteroidales and Clostridiales dominated the gut of high-responders

Among 40 bacterial species whose abundance were higher in HR, 20 (50%) belonged to the Bacteroidales order (Table 6, Fig. S8A). They included 9 Bacteroides species and 7 Prevotella species. Seven species (18%) of the Clostridiales order and 5 Megamonas species (13%) were also observed to dominate the gut of the HR at baseline.

Enterobacterales and Lactobacillales dominated the gut of low-responders

On the other hand, 97 species had lower abundances in the guts of HR as compared to LR dogs (Table 6, Fig. S8A). Forty seven of these species (48%) were in the Enterobacterales order. Species in the Lactobacillales order were also underrepresented in the guts of HR and accounted for 35% of the 97 species. It is worth noting that the abundances of many Enterobacterales and Lactobacillales species increased as a result of diet change when all subjects were included in the analysis (Table 4).

Given the enrichment of Enterobacterales and Lactobacillales in LR as compared to HR, correlative analysis was performed in all dogs. The read counts and the relative abundances of total Enterobacteriales were negatively correlated with the magnitude of PCo1 change (read count: Spearman’s ρ = −0.74, p = 6.66E−06, relative abundance: Spearman’s ρ = −0.69, p = 6.77E−05). Significant correlation was not observed with total Lactobacillales.

Changes in the gut microbiome abundance from baseline to week 4 in high- and low-responders

Not only was the GM of HR and LR different at baseline, HR and LR also responded differently to the diet transition (Table S4, Figs. 7A and 7B).

Enterobacterales and Lactobacillales increased predominantly in high-responders

Over the 4 week period in HR the abundances of 97 species of Enterobacterales increased and none decreased (Table S4 and Fig. 7A). Whereas in LR no increase in abundance of any Enterobacterales species was observed, but rather seven Enterobacterales species were reduced (Fig. 7B). Similarly, the abundances of 77 species in the Lactobacillales order significantly increased and none decreased at week 4 in HR, whereas only five species of Lactobacillales increased and one decreased in LR.

Clostridiales and Bacteroidales had distinctive responses in high- vs. low-responders

A distinctive pattern of change in the Clostridiales and Bacteroidales orders was also noted between HR and LR. In HR, the abundances of 17 species of Clostridiales were reduced whereas there were increased abundances of five species (three of which were members of the genus Clostridium) (Table S4 and Fig. 7A). In LR the abundances of only three species of Clostridiales increased and one species decreased (Fig. 7B). Additionally, the abundance of any Bacteroidales did not significantly change in LR, while seven species of Bacteroidales (six of which were in the genus Prevotella) were reduced in HR.

Gut microbiome relative abundance at week 4 in high- and low-responders

Consistent with a distinctive GM profile at baseline and differential changes between HR and LR, the microbial profiles of HR and LR at week 4 also differed (Table 7, Fig. 6B). A summary of overall GM profile and changes in the abundance of Bacteroidales, Clostridiales, Lactobacillales, and Proteobacteria is also provided in Table 8.

Table 7:

Bacterial species with differential abundances (|fold change| ≥ 2 and p < 0.05) between high-responders (HR, n = 9) and low-responders (LR, n = 10) at week 4.

Phylum	Class	Order	Family	Genus	Species	Relative abundance, in % Median (IQR)		Week 4 HR vs LR
						HR (n = 9)	LR (n = 10)	Log 2 FC mean ± SE	Adjusted P−value1
Higher in HR (68 species)
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Hafnia	alvei	1.88E−03 (1.47E−03 to 1.06E+00)	4.81E−05 (0.00E+00 to 6.65E−05)	10.37 ± 2.02	1.56E−04
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Hafnia	no data	3.57E−04 (1.66E−04 to 1.54E−01)	0.00E+00 (0.00E+00 to 3.58E−05)	10.30 ± 2.26	7.33E−04
Proteobacteria	Gammaproteobacteria	Enterobacterales	Morganellaceae	Morganella	sp HMSC11D09	0.00E+00 (0.00E+00 to 4.86E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	9.07 ± 2.50	8.16E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Hafnia	sp HMSC23F03	3.96E−03 (2.77E−05 to 2.24E−02)	0.00E+00 (0.00E+00 to 1.06E−04)	7.84 ± 2.12	7.29E−03
Firmicutes	Bacilli	Bacillales	Listeriaceae	Listeria	monocytogenes	3.24E−05 (0.00E+00 to 2.65E−04)	0.00E+00 (0.00E+00 to 5.61E−05)	7.67 ± 2.27	1.43E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Solobacterium	moorei	0.00E+00 (0.00E+00 to 1.95E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	7.64 ± 2.77	4.71E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	sp MGH 7	0.00E+00 (0.00E+00 to 5.95E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	7.46 ± 2.68	4.51E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	sp AA405	0.00E+00 (0.00E+00 to 1.33E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	7.36 ± 2.59	4.18E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Cronobacter	dublinensis	1.94E−04 (1.47E−04 to 9.28E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	7.28 ± 1.56	5.73E−04
Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	sp Ep R1	0.00E+00 (0.00E+00 to 6.63E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	7.22 ± 2.61	4.71E−02
Firmicutes	Bacilli	Bacillales	Paenibacillaceae	Paenibacillus	sp VT-16-81	1.19E−04 (0.00E+00 to 3.01E−03)	0.00E+00 (0.00E+00 to 4.34E−05)	7.11 ± 1.97	8.34E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Edwardsiella	hoshinae	2.08E−05 (0.00E+00 to 5.27E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	6.91 ± 2.13	2.12E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	sp CL-2	3.97E−04 (5.54E−05 to 1.66E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	6.70 ± 1.56	1.70E−03
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	tertium	6.77E−05 (0.00E+00 to 7.83E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	6.65 ± 2.01	1.83E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp HMSC072H05	2.65E−04 (0.00E+00 to 7.83E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	6.59 ± 1.84	9.15E−03
Firmicutes	Clostridia	Clostridiales	Ruminococcaceae	Ruminiclostridium	josui	0.00E+00 (0.00E+00 to 1.81E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	6.37 ± 2.37	4.97E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	no data	1.06E−02 (7.68E−03 to 1.80E−01)	1.36E−03 (3.94E−04 to 6.31E−03)	6.14 ± 1.39	1.08E−03
Firmicutes	Bacilli	Lactobacillales	Carnobacteriaceae	Carnobacterium	maltaromaticum	1.08E−03 (2.66E−05 to 3.65E−03)	1.31E−04 (5.01E−05 to 4.23E−04)	6.06 ± 1.54	4.09E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	avium	1.77E−04 (0.00E+00 to 2.88E−02)	0.00E+00 (0.00E+00 to 4.11E−04)	5.99 ± 2.00	3.24E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp KTE11	2.54E−03 (1.06E−03 to 6.28E−03)	0.00E+00 (0.00E+00 to 6.79E−05)	5.94 ± 1.50	4.09E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Erwiniaceae	Pantoea	ananatis	4.52E−04 (2.28E−04 to 6.10E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	5.91 ± 1.56	6.66E−03
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	sp 7 2 43FAA	1.62E−04 (0.00E+00 to 7.34E−04)	0.00E+00 (0.00E+00 to 3.58E−05)	5.88 ± 1.87	2.46E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	minor	3.48E−03 (5.75E−04 to 6.97E−03)	9.53E−04 (1.21E−04 to 3.04E−03)	5.77 ± 1.43	3.54E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	sp MS 92-3	3.97E−05 (0.00E+00 to 2.21E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	5.72 ± 2.03	4.28E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	no data	6.10E−03 (5.07E−03 to 1.04E−02)	1.36E−03 (3.72E−04 to 3.53E−03)	5.64 ± 1.16	3.04E−04
Firmicutes	Clostridia	Clostridiales	Peptostreptococcaceae	Terrisporobacter	glycolicus	1.22E−03 (0.00E+00 to 3.90E−03)	0.00E+00 (0.00E+00 to 4.39E−05)	5.57 ± 1.90	3.59E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Erysipelatoclostridium	ramosum	4.70E−03 (1.30E−04 to 2.69E−02)	8.23E−05 (1.32E−05 to 1.54E−03)	5.57 ± 1.62	1.27E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	sartagoforme	9.73E−05 (0.00E+00 to 9.12E−04)	0.00E+00 (0.00E+00 to 2.42E−05)	5.44 ± 1.75	2.66E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Raoultella	no data	2.66E−05 (0.00E+00 to 3.72E−04)	0.00E+00 (0.00E+00 to 5.18E−05)	5.35 ± 1.77	3.10E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	celatum	2.41E−04 (1.45E−04 to 4.40E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	5.22 ± 1.63	2.16E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	paraputrificum	7.33E−03 (1.55E−03 to 3.41E−02)	2.06E−03 (5.15E−04 to 9.22E−03)	5.01 ± 1.20	2.54E−03
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Coprobacillus	sp 3 3 56FAA	3.47E−03 (1.95E−04 to 4.56E−02)	1.39E−03 (2.32E−04 to 4.99E−03)	4.92 ± 1.34	7.51E−03
Firmicutes	Bacilli	Lactobacillales	Leuconostocaceae	Leuconostoc	mesenteroides	2.21E−03 (2.70E−04 to 1.31E−01)	2.38E−04 (4.92E−05 to 1.15E−02)	4.92 ± 1.75	4.28E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Coprobacillus	no data	1.54E−02 (1.10E−03 to 4.62E−02)	5.15E−04 (1.98E−05 to 7.03E−03)	4.91 ± 1.60	2.85E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	quasipneumoniae	3.91E−04 (3.45E−04 to 4.74E−03)	9.33E−05 (0.00E+00 to 2.56E−04)	4.83 ± 1.65	3.59E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Erwiniaceae	Erwinia	gerundensis	1.77E−04 (1.11E−04 to 2.71E−04)	0.00E+00 (0.00E+00 to 4.76E−05)	4.83 ± 1.25	5.40E−03
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Turicibacter	sanguinis	8.27E−04 (5.54E−05 to 1.52E−03)	2.39E−05 (0.00E+00 to 2.95E−04)	4.76 ± 1.63	3.59E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	no data	no data	3.08E−01 (1.35E−02 to 2.33E+00)	1.07E−01 (1.37E−02 to 1.31E−01)	4.73 ± 1.28	7.46E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Raoultella	ornithinolytica	1.86E−04 (4.89E−05 to 3.96E−03)	3.45E−05 (0.00E+00 to 1.38E−04)	4.68 ± 1.67	4.40E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	no data	9.48E−03 (8.22E−03 to 7.28E−02)	1.61E−03 (2.52E−04 to 8.19E−03)	4.61 ± 1.41	2.02E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp KTE172	1.67E−02 (9.42E−03 to 1.64E−01)	4.98E−03 (8.28E−04 to 1.45E−02)	4.57 ± 1.29	9.85E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp 1 1 43	1.57E−02 (5.90E−03 to 2.83E−02)	1.07E−03 (3.16E−04 to 5.30E−03)	4.54 ± 1.15	4.09E−03
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Turicibacter	no data	1.03E−03 (3.09E−04 to 2.31E−03)	2.11E−04 (0.00E+00 to 1.64E−03)	4.32 ± 1.54	4.35E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Shigella	no data	9.74E−01 (2.15E−01 to 1.97E+00)	4.37E−02 (1.42E−02 to 1.51E−01)	4.29 ± 1.15	7.29E−03
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	pneumoniae	2.68E−03 (9.12E−04 to 1.63E−02)	5.40E−04 (8.12E−05 to 1.14E−03)	4.09 ± 1.36	3.19E−02
Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Acinetobacter	indicus	1.14E−03 (1.33E−04 to 1.81E−03)	0.00E+00 (0.00E+00 to 1.26E−04)	3.99 ± 1.48	4.97E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	sp KTE96	1.83E−03 (4.83E−04 to 3.30E−03)	9.68E−05 (0.00E+00 to 7.69E−04)	3.98 ± 1.45	4.76E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	freundii complex sp CFNIH2	2.13E−04 (1.66E−04 to 7.21E−04)	4.00E−05 (0.00E+00 to 9.02E−05)	3.94 ± 1.45	4.81E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	cloacae	1.49E−03 (3.42E−04 to 1.95E−03)	1.08E−04 (0.00E+00 to 3.00E−04)	3.92 ± 1.23	2.26E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Shigella	flexneri	3.42E−01 (2.39E−01 to 7.99E−01)	4.10E−02 (7.24E−03 to 1.49E−01)	3.90 ± 1.14	1.30E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Siccibacter	turicensis	1.96E−04 (1.33E−04 to 2.08E−04)	4.84E−05 (0.00E+00 to 1.21E−04)	3.89 ± 1.12	1.21E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	no data	bacterium strain FGI 57	3.05E−03 (9.31E−04 to 8.49E−03)	7.14E−04 (1.58E−04 to 1.31E−03)	3.86 ± 1.20	2.15E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	freundii	5.34E−03 (9.58E−04 to 2.11E−02)	6.44E−04 (2.16E−04 to 1.19E−03)	3.83 ± 1.42	4.97E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Coprobacillus	sp 8 2 54BFAA	5.63E−03 (5.86E−04 to 2.25E−02)	2.28E−03 (2.22E−04 to 5.50E−03)	3.72 ± 1.15	2.15E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Yokenella	regensburgei	5.32E−04 (3.33E−04 to 9.28E−04)	9.74E−05 (4.14E−05 to 2.38E−04)	3.71 ± 1.16	2.16E−02
Proteobacteria	Gammaproteobacteria	no data	no data	no data	no data	2.66E−02 (6.47E−03 to 1.03E−01)	2.62E−03 (7.30E−04 to 9.95E−03)	3.70 ± 1.29	4.11E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	werkmanii	3.72E−04 (5.32E−05 to 2.93E−03)	5.87E−05 (3.61E−05 to 2.19E−04)	3.63 ± 1.20	3.19E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Shigella	sonnei	2.11E−01 (1.90E−01 to 4.21E−01)	2.15E−02 (5.35E−03 to 1.19E−01)	3.58 ± 1.15	2.66E−02
Actinobacteria	Actinobacteria	Corynebacteriales	Corynebacteriaceae	Corynebacterium	striatum	5.55E−04 (4.42E−04 to 6.65E−04)	1.20E−04 (1.73E−05 to 2.52E−04)	3.46 ± 1.02	1.40E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	no data	no data	2.40E+01 (1.57E+01 to 3.93E+01)	3.26E+00 (3.15E−01 to 1.05E+01)	3.34 ± 1.17	4.11E−02
Actinobacteria	Actinobacteria	Corynebacteriales	Nocardiaceae	Rhodococcus	opacus	6.19E−04 (4.44E−04 to 7.18E−04)	7.17E−05 (0.00E+00 to 3.01E−04)	3.32 ± 1.13	3.53E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Coprobacillus	sp D7	3.57E−03 (4.86E−04 to 1.51E−02)	2.00E−03 (4.98E−04 to 5.34E−03)	3.29 ± 1.15	4.11E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Escherichia	albertii	3.53E−01 (1.71E−01 to 5.09E−01)	6.62E−02 (1.44E−02 to 1.43E−01)	3.29 ± 1.15	4.16E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Citrobacter	sp MGH110	1.66E−04 (1.45E−04 to 6.09E−04)	6.33E−05 (0.00E+00 to 1.34E−04)	3.26 ± 1.15	4.28E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Kluyvera	cryocrescens	1.04E−03 (7.06E−04 to 1.46E−03)	1.46E−04 (8.05E−06 to 5.91E−04)	3.25 ± 1.09	3.39E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Enterobacter	no data	5.65E−03 (4.07E−03 to 8.89E−03)	7.39E−04 (1.65E−04 to 2.59E−03)	3.09 ± 1.13	4.81E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Kosakonia	cowanii	4.42E−04 (3.17E−04 to 4.79E−04)	5.90E−05 (0.00E+00 to 3.21E−04)	2.99 ± 1.10	4.90E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Shigella	boydii	2.11E−01 (7.81E−02 to 3.71E−01)	2.20E−02 (6.29E−03 to 7.84E−02)	2.90 ± 1.07	4.90E−02
Lower in HR (12 species)
Actinobacteria	Coriobacteriia	Eggerthellales	Eggerthellaceae	Eggerthella	no data	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 9.63E−05)	−8.77 ± 2.94	3.39E−02
Actinobacteria	Actinobacteria	Bifidobacteriales	Bifidobacteriaceae	Bifidobacterium	gallinarum	0.00E+00 (0.00E+00 to 1.47E−04)	0.00E+00 (0.00E+00 to 1.97E−02)	−7.36 ± 2.70	4.81E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Anaerostipes	caccae	0.00E+00 (0.00E+00 to 0.00E+00)	2.42E−05 (0.00E+00 to 4.28E−03)	−6.45 ± 2.38	4.90E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Anaerostipes	no data	2.08E−05 (0.00E+00 to 1.33E−04)	1.23E−04 (8.06E−06 to 4.72E−02)	−6.20 ± 2.01	2.76E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	canintestini	7.98E−05 (0.00E+00 to 2.92E−04)	3.40E−04 (3.54E−05 to 1.04E−02)	−5.57 ± 1.62	1.27E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Catenibacterium	mitsuokai	8.32E−05 (0.00E+00 to 7.59E−04)	2.97E−02 (1.35E−03 to 5.00E−01)	−5.29 ± 1.71	2.66E−02
Actinobacteria	Actinobacteria	Bifidobacteriales	Bifidobacteriaceae	Bifidobacterium	no data	0.00E+00 (0.00E+00 to 2.22E−04)	3.54E−04 (3.63E−05 to 1.07E−02)	−4.80 ± 1.75	4.76E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	pasteurianus	4.89E−04 (1.38E−04 to 3.65E−03)	6.72E−03 (1.91E−03 to 2.25E−01)	−4.26 ± 1.55	4.76E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Anaerostipes	sp 3 2 56FAA	1.55E−03 (2.92E−04 to 1.01E−02)	2.29E−02 (5.94E−03 to 4.40E−02)	−3.96 ± 1.07	7.29E−03
Firmicutes	Clostridia	Clostridiales	Eubacteriaceae	no data	bacterium CHKCI004	0.00E+00 (0.00E+00 to 3.97E−05)	1.54E−04 (5.61E−05 to 5.19E−04)	−3.63 ± 1.23	3.48E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	no data	bacterium 3 1 46FAA	5.06E−04 (1.35E−04 to 3.67E−03)	5.23E−03 (2.61E−03 to 3.45E−02)	−2.85 ± 1.03	4.71E−02
Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	sp SS2/1	2.93E−04 (6.49E−05 to 6.85E−04)	1.51E−03 (7.01E−04 to 4.71E−03)	−2.47 ± 0.85	3.59E−02

DOI: 10.7717/peerj.11648/table-7

Notes:

1 P values were adjusted with false discovery rate for multiple comparisons.

FC, fold change; SE, standard error.

Table 8:

Summary of the differential abundances of Bacteroidales, Clostridiales, Lactobacillales, and Enterobacterales at baseline and week 4 in all dogs (n = 28), high- (n = 9), and low-responders (n = 10).

Bacteria (Order)	Subjects	Baseline	Change in the abundance	Week 4
Bacteroidales 96 species compared	All	–	↑1 species (1.0%) ↓5 species (5.2%)	–
	HR	HR>LR: 20 species (20.8%) HR<LR: 0 species (0%)	↓7 species (7.3%)	HR>LR: 0 species (0%) HR<LR: 0 species (0%)
	LR		↑1 species (1.0%)
Clostridiales 205 species compared	All	–	↑3 species (1.5%) ↓13 species (6.3%)	–
	HR	HR>LR: 7 species (3.4%) HR<LR: 3 species (1.5%)	↑5 species (2.4%) ↓17 species (8.3%)	HR>LR: 9 species (4.4%) HR<LR: 6 species (2.9%)
	LR		↑3 species (1.5%) ↓1 species (0.5%)
Lactobacillales 205 species compared	All	–	↑47 species (22.9%) ↓2 species (1.0%)	–
	HR	HR<LR: 34 species (16.6%) HR>LR: 1 species (0.5%)	↑77 species (37.6%)	HR>LR: 5 species (2.4%) HR<LR: 2 species (1.0%)
	LR		↑5 species (2.4%) ↓1 species (0.5%)
Enterobacteriales 205 species compared	All	–	↑17 species (8.3%)	–
	HR	HR<LR: 47 species (22.9%) HR>LR: 0 species (0%)	↑97 species (47.3%)	HR<LR: 0 species (0%) HR>LR: 38 species (18.5%)
	LR		↓7 species (3.4%)

DOI: 10.7717/peerj.11648/table-8

Notes:

Data expressed as numbers of species and percentages of all species being compared in each order.

Enterobacterales and Lactobacillales dominated the gut of high-responders

Among 68 bacterial species whose abundances were higher in HR, 38 (56%) were Enterobacterales (Table 7, Fig. S8B). No Enterobacterales species was observed to be higher in LR at week 4. This was the opposite of baseline GM where 47 species of Enterobaterales were higher in LR (Table 6). For Lactobacillales, five species were enriched in the gut of HR while two species were enriched in the gut of LR at week 4.

Clostridiales in the gut of high- and low-responders

Nine (13%) out of 68 species with higher abundances among HR were in the Clostridiales order, while six (50%) out of 12 species with higher abundances among LR were in the Clostridiales order (Table 7, Fig. S8B).

Bacteroidales was no longer different in high- vs low-responders

The 20 species in the Bacteroidales order observed to have higher abundances in HR at baseline were no longer different between HR and LR at week 4 (Table 7, Fig. S8B).

Protein content of the baseline diet associated with the magnitude of PCo1 change

In addition to baseline GM, the magnitude of PCo1 change was also associated with baseline diet composition. Data on the baseline extruded kibble diets were assembled; however, guaranteed nutrient analysis could only be determined for 15 dogs whose specific formula was provided (Table S5). Protein content of the baseline diet statistically differed among HR, MR, and LR, when expressed as either % dry matter (p = 0.040) or gram per 1,000 kcal diet (p = 0.027, Kruskal–Wallis test). In addition, % protein dry matter was also negatively correlated with PCo1 score at baseline (Spearman’s ρ = −0.54, p = 0.039) (Fig. S9A), and positively correlated with PCo1 score change over the 4 week (Spearman’s ρ = 0.67, p = 0.006) (Fig. S9B). A similar finding was seen when protein content was expressed as g/1000 kcal (Spearman’s ρ = −0.59, p = 0.024 for correlation with PCo1 score at baseline; Spearman’s ρ = 0.78, p = 9.9E−04 for correlation with PCo1 score change) (Figs. S9C, S9D). No association with PCo1 score was observed for fat, fiber, or calorie density. Changes in PCo1 score were also not associated with any of the four different recipes of the intervention diet.

Subject characteristics and α-diversity in high-, mid- and low-responders

Other subject characteristics among the HR, MR, and LR are summarized in Table S6A. Age, sex, body weight, BCS and other subject characterisitics at baseline were not significantly different among the groups. Likewise, health outcomes reported at week 4 were also not associated with the magnitude of PCo1 score change (Table S6B). Additionally, no measure of α-diversity was different among HR, MR, and LR at baseline or at week 4 at a sequencing coverage of 470,000 reads (Table S7).

Gut microbiome and change in overall health

To investigate the association between changes in the microbial profile and overall health, the GM of dogs with reported “better” (n = 19) and “no change” (n = 12) in overall health were compared at the species level at week 4 (Tables 3, 9, Fig. 8). Dogs whose owners reported improved health had higher abundances of 35 species, 24 (69%) of which are in the Lactobacillales order and two of which are in the Enterobacterales order. Five (14%) species of the Erysipelotrichaceae family were also higher in dogs reported as having better overall health. Two species, both Bacteroides, were lower in those reported as having better overall health.

Table 9:

Bacterial species whose abundance at week 4 was significantly different (|fold change| ≥ 2 and FDR-adjusted p < 0.05) between those reporting “better” (n = 16) and “about the same” overall health (n = 10) after diet change.

Phylum	Class	Order	Family	Genus	Species	Relative abundance, in % Median (IQR)		Better vs About the same health Week 4
						Better overall health (n = 16)	About the same overall health (n = 10)	Log 2 FC mean ± SE	Adjusted P-value1
Higher in “better overall health” (35 species)
Firmicutes	Bacilli	Bacillales	Bacillaceae	Lysinibacillus	sphaericus	0.00E+00 (0.00E+00 to 0.00E+00)	0.00E+00 (0.00E+00 to 0.00E+00)	25.39 ± 3.04	3.41E−14
Actinobacteria	Actinobacteria	Bifidobacteriales	Bifidobacteriaceae	Bifidobacterium	animalis	0.00E+00 (0.00E+00 to 2.94E−05)	0.00E+00 (0.00E+00 to 0.00E+00)	10.52 ± 3.04	1.54E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	avium	1.85E−04 (0.00E+00 to 9.98E−04)	0.00E+00 (0.00E+00 to 3.11E−05)	8.40 ± 2.03	3.09E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp 3C7 DIV0644	6.49E−05 (0.00E+00 to 2.20E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	8.39 ± 1.94	2.71E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	plantarum	3.25E−04 (8.20E−05 to 1.99E−03)	1.18E−04 (0.00E+00 to 1.64E−03)	8.35 ± 1.68	1.84E−04
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp 3G6 DIV0642	8.56E−04 (0.00E+00 to 6.13E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	7.89 ± 1.84	2.71E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp HMSC072H05	1.33E−05 (0.00E+00 to 2.88E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	7.87 ± 2.36	1.88E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp FDAARGOS 163	1.35E−04 (0.00E+00 to 3.44E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	7.30 ± 1.95	6.49E−03
Actinobacteria	Actinobacteria	Micrococcales	Micrococcaceae	Arthrobacter	no data	8.09E−05 (0.00E+00 to 3.75E−04)	0.00E+00 (0.00E+00 to 3.29E−05)	7.05 ± 1.83	5.68E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp 5B3 DIV0040	4.23E−04 (0.00E+00 to 5.17E−03)	0.00E+00 (0.00E+00 to 2.42E−05)	6.66 ± 1.64	3.09E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp CR-Ec1	7.63E−05 (0.00E+00 to 1.59E−03)	0.00E+00 (0.00E+00 to 0.00E+00)	6.39 ± 1.85	1.54E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp kppr-6	5.33E−05 (0.00E+00 to 4.47E−03)	0.00E+00 (0.00E+00 to 2.42E−05)	6.17 ± 1.63	6.25E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp FDAARGOS 375	9.02E−04 (2.08E−05 to 1.91E−02)	4.83E−05 (0.00E+00 to 2.76E−04)	6.12 ± 1.50	3.09E−03
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	orisratti	1.02E−04 (0.00E+00 to 7.83E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	5.94 ± 1.88	3.03E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	casseliflavus	4.22E−03 (3.18E−04 to 5.93E−02)	5.53E−05 (6.27E−06 to 4.45E−04)	5.68 ± 1.49	5.79E−03
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	brevis	2.64E−04 (0.00E+00 to 8.82E−04)	5.09E−05 (3.45E−05 to 5.27E−04)	5.61 ± 1.49	6.49E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	pseudoavium	7.17E−05 (0.00E+00 to 3.14E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	5.49 ± 1.57	1.51E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Hafniaceae	Hafnia	alvei	8.40E−04 (5.64E−05 to 1.16E−01)	3.45E−05 (0.00E+00 to 4.21E−04)	5.48 ± 1.60	1.60E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Catenibacterium	mitsuokai	4.69E−03 (3.99E−05 to 7.00E−01)	7.95E−04 (1.71E−04 to 2.00E−02)	5.46 ± 1.69	2.62E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp 6D12 DIV0197	2.71E−04 (0.00E+00 to 9.29E−03)	0.00E+00 (0.00E+00 to 6.22E−05)	5.38 ± 1.81	4.60E−02
Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Raoultella	ornithinolytica	1.96E−04 (0.00E+00 to 3.51E−03)	5.90E−05 (8.27E−06 to 1.22E−04)	5.34 ± 1.34	3.73E−03
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	sp HMSC05C03	1.11E−04 (0.00E+00 to 8.99E−04)	0.00E+00 (0.00E+00 to 0.00E+00)	5.28 ± 1.58	1.87E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	canis	1.46E−04 (0.00E+00 to 1.36E−03)	3.45E−05 (0.00E+00 to 1.93E−04)	5.11 ± 1.67	3.78E−02
Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Anaerostipes	no data	1.82E−04 (0.00E+00 to 2.27E−02)	4.25E−05 (5.19E−06 to 3.35E−03)	4.83 ± 1.57	3.63E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	gallinarum	1.84E−03 (2.06E−04 to 8.66E−03)	8.58E−05 (0.00E+00 to 1.66E−03)	4.82 ± 1.34	1.04E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	dispar	7.80E−05 (0.00E+00 to 1.00E−03)	0.00E+00 (0.00E+00 to 6.09E−05)	4.75 ± 1.45	2.23E−02
Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	malodoratus	1.66E−04 (3.32E−05 to 5.32E−04)	0.00E+00 (0.00E+00 to 3.67E−05)	4.72 ± 1.38	1.64E−02
Firmicutes	Bacilli	Lactobacillales	Carnobacteriaceae	Carnobacterium	maltaromaticum	2.01E−04 (9.79E−05 to 3.96E−03)	2.93E−05 (0.00E+00 to 9.28E−04)	4.30 ± 1.36	3.03E−02
Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	fermentum	3.96E−04 (0.00E+00 to 1.01E−03)	6.09E−05 (0.00E+00 to 2.59E−04)	4.29 ± 1.37	3.14E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Lactococcus	garvieae	4.12E−04 (1.69E−04 to 1.80E−03)	1.88E−04 (0.00E+00 to 4.24E−04)	4.09 ± 1.21	1.74E−02
Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Lactococcus	lactis	2.18E−02 (2.76E−03 to 1.32E−01)	6.68E−03 (3.89E−03 to 2.35E−02)	3.88 ± 1.12	1.54E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Erysipelatoclostridium	innocuum	6.12E−04 (2.26E−04 to 3.63E−03)	1.46E−04 (3.54E−05 to 2.50E−04)	3.83 ± 0.94	3.09E−03
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Coprobacillus	sp 3 3 56FAA	4.90E−03 (8.45E−04 to 1.62E−02)	8.80E−04 (1.98E−05 to 3.02E−03)	3.54 ± 1.13	3.14E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	no data	no data	2.01E−01 (1.77E−02 to 1.16E+00)	2.66E−02 (4.94E−03 to 2.53E−01)	3.35 ± 1.09	3.63E−02
Firmicutes	Erysipelotrichia	Erysipelotrichales	Erysipelotrichaceae	Coprobacillus	sp 8 2 54BFAA	6.89E−03 (2.25E−03 to 1.77E−02)	5.31E−04 (5.01E−05 to 4.51E−03)	3.15 ± 1.06	4.60E−02
Lower in “better overall health” (2 species)
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	stercoris	7.21E−05 (0.00E+00 to 1.05E−03)	1.43E−03 (0.00E+00 to 1.59E−01)	−6.79 ± 1.71	3.73E−03
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	coprocola	1.50E−05 (0.00E+00 to 5.17E−04)	2.17E−04 (2.38E−05 to 5.16E−02)	−6.58 ± 1.60	3.09E−03

DOI: 10.7717/peerj.11648/table-9

Notes:

1 P values were adjusted with false discovery rate for multiple comparisons.

FC, fold change; SE, standard error; NS, not significant.

Since the overall health status at baseline was comparable between these two groups of dogs, as a control the same analysis was performed on the sample collected at baseline. Only 8 species were identified to be differentially present between the two groups (Table S8). None of the identified species were a Lactobacillales, an Enterobacterales, or an Erysipelotrichaceae.

The magnitude of change in PCo1 score was not significantly different between dogs reported as having improved versus no change in overall health. Species evenness, richness, and Shannon’s diversity index were not different between these two groups at baseline at the sequencing coverage of 470,000 reads. At week 4, however, the species evenness in those reporting improved overall health was significantly higher than those reporting no change in overall health (0.59 ± 0.10 vs 0.52 ± 0.11, p = 0.023, Kruskal–Wallis test). Likewise, Shannon’s diversity index at week 4 was higher in those reported to have improved overall health (2.39 ± 0.39 vs 2.10 ± 0.44, p = 0.023, Kruskal–Wallis test).

Discussion

We describe GM composition and owner-reported health perception of dogs switching from various brands of kibble to commercial MC diets. To our knowledge, this is the first MC diet study performed in household dogs, including male and female dogs of various breeds across a broad age range. As a result of this diverse and free-living study population these findings may be more generalizable to the dog population than other studies that focus on a single breed or a single diet. Two previously published studies using a similar diet comparison were conducted, however they consisted of eight and 12 Beagles in a laboratory setting (Algya et al., 2018; Do et al., 2021). Dogs in the present study maintained their body weight throughout the study period, therefore changes in health and GM were independent of weight change. Finally, the characterization of gut bacteria in this study was performed with metagenomic sequencing, a powerful technique enabling enhanced taxonomic resolution and accuracy, as well as increased detection of diversity and functional genes, a technique that has not yet been widely used in canine research (Ranjan et al., 2016; Salas-Mani et al., 2018; Xu et al., 2019; Alessandri et al., 2019; Connelly et al., 2019; Do et al., 2021).

All dogs were perceived by their owners as having either better or no change in overall health after diet transition. It is possible that this observation could be subject to bias and may not directly correlate to more objective health outcomes, but as argued by Yeates & Main (2009), owner-reported health assessment is a separate and independent outcome that is especially important in this type of research study. A chance of bias may also derive from the willingness of participants to report the health impact associated with the diet transition, and the observation that a significant portion of participants (24%) did not complete the study. However the association between a MC diet and positive health perception has been previously reported using a survey administered to a group of dog owners after a veterinarian formulated MC diets for the dogs (Johnson et al., 2016). Dogs accepted MC diets well and remained healthy in two additional feeding trials (Algya et al., 2018; Do et al., 2021).

Incidents of vomiting and diarrhea were recorded over the four weeks after diet transition. Currently there is not much data on the prevalence and frequency of these symptoms in household dogs, regardless of diet transition. A 2005 survey collecting responses from 772 dog owners in England found that the rate of spontaneous vomiting and diarrhea was one case per 10.6 dog-weeks and one case per 13.4 dog-weeks, respectively (Hubbard et al., 2007). A diet change was suggested to be a cause of diarrhea in some dogs. These are slightly higher than the rates in this present study. Likewise, 27% of dogs experienced diarrhea within seven weeks after boiled minced beef replaced 75% of the calories in an extruded diet (one case per 25.6 dog-weeks) (Herstad et al., 2017, 2018). Other diet transition studies using a raw or MC diet did not report cases of diarrhea or vomiting (Sandri et al., 2017; Algya et al., 2018; Do et al., 2021). Therefore, the rates of vomiting and diarrhea appear to be consistent with data available from other household dog populations.

Fifty eight percent of owners reported lower defecation frequency upon diet transition in this study, although this proportion was not significantly different from those reporting no change. Fecal output has been reported to be significantly lower in dogs fed MC diets compared to extruded diets, despite dogs maintaining the same body weight on different diets (Oba et al., 2020; Do et al., 2021). The extremely high nutrient and energy digestibility of MC diets is suggested to be the main driver of the reduction in fecal output (Do et al., 2021). There was also a non-significant trend towards firmer stool when dogs switched to the test diets in our study. In studies using veterinarian-assessed data or an objective analysis of fecal moisture, fecal consistency was reported to be firmer (Sandri et al., 2017; Bermingham et al., 2017) or comparable (Algya et al., 2018; Schmidt et al., 2018; Do et al., 2021) among healthy dogs fed MC or raw diets compared to those fed a traditional extruded diet.

The intervention diet in this study consisted of many nutrients and dietary components that may have an influence on both health and GM composition, including water (Cameron et al., 2011; Deng et al., 2014; Alexander, Colyer & Morris, 2014), prebiotic fibers (Middelbos et al., 2010; Panasevich et al., 2015; Alexander et al., 2018; Eisenhauer et al., 2019), omega-3 fatty acids (Bauer, 2011), carotenoids, and polyphenols (Kolchin et al., 1991; Kim et al., 2000; Chew et al., 2000; Massimino et al., 2003; Hall et al., 2006; Carrera-Quintanar et al., 2018; Lyu et al., 2018), although high-quality extruded diets may include these nutrients and compounds as well. Nutrient and energy digestibility has also been observed to be different between extruded kibble and MC diets (Algya et al., 2018), and it may facilitate differential impacts on health and GM. Due to the study design, it is impossible to pinpoint which component(s) of the MC diets contributed to the change in health and GM profile. Similarly, the four MC dietrecipes were different in nutrient and in ingredient composition, adding an additional confounding factor.

Changes in α-diversity in response to dietary interventions have varied between studies, likelybecause they are dependent on the diets being compared. Similar to Do et al. (2021), we did not observe any change in the GM α-diversity between kibble and MC diets. A cross-sectional study performed in household dogs also reported no differences in α-diversity between those fed raw and extruded diets (Schmidt et al., 2018). However these findings contrast with previous reports that demonstrate reduced α-diversity upon transitioning to a MC diet in 8 or 11 dogs (Herstad et al., 2017; Algya et al., 2018), or increased α-diversity upon transitioning to a raw food diet in eight dogs (Sandri et al., 2017). α-Diversity was also higher in six household dogs fed raw diets as compared to five household dogs fed commercial extruded diets (Kim et al., 2017). Herstad et al. argued that decreased α-diversity with a cooked beef diet may be due to higher fat content inducing bile production (Herstad et al., 2017), which has been shown to be antimicrobial (Yokota et al., 2012; Herstad et al., 2017). As the fat and protein content in the baseline extruded diets were not statistically different from the intervention diet in this present study, it is possible that the lack of macronutrient composition difference is responsible for the lack of a change in α-diversity. The variety of baseline diets and the utilization of four different MC recipes may also mask the effect of diet transition on α-diversity. Often, the α-diversity is observed to be lower in diseased populations (Wernimont et al., 2020). In this study the Shannon’s diversity index in a subgroup of dogs whose overall health reported to be improved at week four was significantly higher than those that remained unchanged. However, the use of α-diversity and its clinical importance have been recently challenged (Moya & Ferrer, 2016).

Consistent with previous shotgun metagenomic sequencing studies in household dogs (Alessandri et al., 2019; Jha et al., 2020), four phyla, Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes, dominated the GM. A variety of taxonomic differences in the microbiome were observed after dietary transition, including increased abundances of many LAB. The abundances of the Lactobacillaceae and Leuconostocaceae families and the Lactobacillus genus were similarly reported to be higher in dogs fed a raw diet compared to a kibble diet (Bermingham et al., 2017; Schmidt et al., 2018). On the contrary, Do et al. (2021) reported lower levels of Lactobacillus in dogs fed MC diets compared to an extruded kibble, but the kibble contained an L. acidophilus fermentation product and high chicory content which were postulated to contribute to the growth of LAB. In many mammals LAB have been suggested to be allochthonous, meaning they are ingested through their natural occurrence on food rather than being true habitants of the distal part of the host GI tract (Walter, 2008). Owing to their demonstrated ability to improve GI health (Grześkowiak et al., 2015; Jensen & Bjørnvad, 2019), selective LAB are often included in probiotic food or supplements. We also observed a significant association between increased abundances of Lactobacillales and dogs reported as having better health after four weeks of the intervention diet.

An increase of Enterobacterales was also observed with diet transition. Similar increases were previously observed in healthy dogs fed either a MC or a raw diet (Sandri et al., 2017; Algya et al., 2018; Schmidt et al., 2018). While there are pathogens within this order, many species of Enterobacterales, such as Salmonella and E. coli, are also members of the healthy human and canine GM, and only opportunistically cause disease in humans, for instance, when an immune system is compromised (Moon et al., 2018). Frequently, only specific strains are pathogenic. It is unknown whether some species of Enterobacterales are also associated with increased disease risk in dogs, particularly since some known human pathogens (e.g. Escherichia/Shigella spp. (Sandri et al., 2017), β-lactamase producing E. coli (Baede et al., 2015), Salmonella (Reimschuessel et al., 2017), and Campylobacter spp. (Bojanić et al., 2017)) are present in the GI tract of healthy dogs. Additionally, Enterobacterlaes are diverse and have a considerably wide range of abundances in the present study. A wide range of Proteobacteria, whose members predominantly include Enterobacterales, have been previously reported in other cohorts of healthy dogs using metagenomic sequencing (IQR: 3–52% in (Jha et al., 2020)) or 16S rRNA sequencing (range: 0–22% in (Hand et al., 2013), range: 0–17% in (Garcia-Mazcorro et al., 2012)). Therefore the impact of an increased abundance of Enterobacterales on canine health remains to be determined.

Switching to the MC diet also resulted in reduced abundances of some taxa, including species in the order Clostridiales and certain genera, including Blautia, Prevotella, Peptostreptococcus, and Faecalibacterium. This result shares some similarities with previous reports with a transition from an extruded kibble to a raw diet (Sandri et al., 2017; Bermingham et al., 2017; Algya et al., 2018; Schmidt et al., 2018). A MC diet also decreased Prevotella in dogs switching from an extruded kibble diet (Do et al., 2021), but not Clostridium, Blautia, or Faecalibacterium. Many of these bacteria are present in a healthy human GI tract, but their relevance is unknown in dogs. F. prausnitzii, which was lower after the intervention diet, was also shown to be lower in dogs fed minced beef than those fed an extruded diet (Herstad et al., 2017). This bacterium is thought to be beneficial because of its ability to produce butyrate, a short-chain fatty acid that serves as an energy source for colonocytes (Louis & Flint, 2009; Donohoe et al., 2011). However, its reduced abundance in MC diets may be due to other butyrate-producing bacteria becoming more abundant (e.g. Firmicutes (Macfarlane & Macfarlane, 2003)), as it has been shown with increased levels of Roseburia and butyrate in dogs fed minced beef (Herstad et al., 2017), but not in the previous trial (Do et al., 2021), or in the present study. This is further supported by the observation that the KO terms related to short-chain fatty acid metabolism remained unchanged upon diet transition in this study.

Our results indicate a differential microbiomic response to dietary transition based on the magnitude of change in β-diversity (i.e. HR, MR and LR). While similar analytical approaches have been adopted in human research (Salonen et al., 2014; Mills et al., 2019), to our knowledge this is the first study to report distinct varied microbial responses in dogs in response to diet change. Using assorted multivariate analyses, the GM at baseline was demonstrated to be different among HR, MR and LR. Specifically, HRs had lower abundances of Lactobacillales and Enterobacterales, and higher abundances of Bacteroidales than the LRs, and they responded to the MC diet differently and more robustly. We hypothesize that the niche occupied by Bacteroidales at baseline becomes more favorable for Enterobacterales and LAB growth upon diet transition. This may explain why the lower abundance of Enterobacterales at baseline was associated with greater PCo1 score change. Moreover, the protein content of the baseline diet was positively correlated with the magnitude of PCo1 change, although we acknowledge the small sample size (n = 15) as well as a previous analysis of 750 commercial dry pet foods demonstrating 1.6% difference between the guaranteed and actual analysis of crude protein (Hill et al., 2009). Given that the protein content of the baseline and the intervention diets were statistically comparable, and that Enterobacterales are able to thrive on proteins, carbohydrates, or lipids (Moon et al., 2018), the observed correlation may be explained by the change in the ingredients despite an unmodified macronutrient profile. For example, replacing chicken with whole cricket meal led to a small change in the GM profile in dogs (Jarett et al., 2019), and replacing cellulose and brewer’s rice with potato fiber up to 6% also led to an increase of Faecalibacterium (Panasevich et al., 2015). Such associative findings between baseline characteristics and magnitude of responses to the intervention can serve as a basis for future studies that aim to construct predictive models, and ultimately, dietary plans tailored to each individual based on existing microbiome composition and function for optimized health outcomes (Zmora et al., 2018).

Changes in GM function characterized by KEGG were detected with a multivariate analysis (PCoA), but remained less defined with univariate analysis (negative binomial GLM with DESeq2) in this study. The univariate method is inferior in terms of its ability to capture small changes cumulatively. GM functional redundancy under different dietary environments has been demonstrated (Moya & Ferrer, 2016), and changes in GM profile in healthy dogs may not reflect a drastic change in the functionality as compared to those with improved or worsened health status after an intervention (Guard et al., 2015). Thus the lack of any drastic change in functionality is likely at least partially reflective of dogs being healthy at both baseline and week 4. Availability of data from other platforms such as transcriptional profiling or targeted metabolomics may complement metagenomic sequencing in capturing finer changes in GM functionality.

In conclusion, we have demonstrated significant changes in the GM profile of healthy dogs transitioning from a variety of kibble diets to MC diets. The magnitude of shift is associated with baseline GM profile and protein content of the baseline diet. The test diets were well accepted throughout the study, and the majority of dog owners report an improvement or no change in specific health outcomes. Despite its novelty, there are some limitations to the study. A control group where dogs remained on the baseline diet for four weeks was not included, thus any direct causal relationship cannot be established between diet transition and outcomes. However, observed changes in the GM were likely to be caused by diet change as minimal changes were expected if baseline diets were maintained for four weeks. While the four different MC diet recipes may have dampened the signal, most dogs were on a mixed-recipe diet and a wide variety of baseline diets may have provided an opportunity to investigate the wide range of diets and responses among subjects. Finally, while no clinical measures were available, the health data do reflect the owner’s perceptions of health.

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