Effects of a synbiotic on the fecal microbiome and metabolomic profiles of healthy research cats administered clindamycin: a randomized, controlled trial

Figures & data

Table 1. Dysbiosis index and alpha diversity results for cats that received clindamycin and placebo or synbiotic.

	Baseline		Days 26-28		Days 68-70
	Placebo	Synbiotic	Placebo^+	Synbiotic	Placebo	Synbiotic	P-value
Dysbiosis index	-3.3^c	-4.7^c	1.2^a	0.6^a	-2.2^b	-3.7^b	<0.01 (time)
	(-5.0 to -2.1)	(-5.5 to -2.2)	(0.8 to 1.6)	(-0.2 to 5.9)	(-4.8 to 0.1)	(-6.2 to -1.7)
Shannon index	4.5^abc	5.7^a	4.0^bc	4.7^b	4.8^abc	4.9^c	0.03 (group by time),
	(3.9-5.8)	(4.6-6.6)	(3.4-4.3)	(3.5-4.8)	(3.4-5.9)	(4.0-5.8)	<0.01 (time)
Goods coverage	0.9843^b	0.9832^b	0.9887^a	0.9860^a	0.9843^b	0.9842^b	<0.01 (time)
	(0.9781-0.9907)	(0.9784-0.9883)	(0.9866-0.9907)	(0.9831-0.9919)	(0.9799-0.9906)	(0.9809-0.9901)
Chao1 metric	2,211^a	2,590^a	1,644^b	1,950^b	2,273^ab	2,169^ab	0.02 (time)
	(1,276-3,242)	(1,645-3,324)	(1,307-1,896)	(1,230-2,546)	(1,243-2,933)	(1,206-2,927)

Median (range) results for feces collected at baseline (days 5–7), at the conclusion of antibiotic administration (days 26–28), and after a 6-week washout (days 68–70) from 16 healthy cats, 8 per group,⁺ that received 75 mg clindamycin followed 1 h later by either two capsules of a placebo or synbiotic PO once daily for 21 days.

⁺Feces not available from four cats at time point 26–28 because of early termination of treatment due to severe gastrointestinal signs. Values that do not share a common superscript letter differed significantly (P < 0.05) based on post hoc analysis.

Figure 1. Principal coordinate analysis (PCoA) of unweighted uniFrac distances of 16S rRNA genes.

Gene sequences were determined using fecal samples collected at baseline (days 5–7), at the conclusion of antibiotic administration (days 26–28), and after a 6-week washout (days 68–70) from 16 healthy cats, 8 per group,⁺ that received 75 mg clindamycin followed 1 h later by either two capsules of a placebo or synbiotic PO once daily for 21 days. ⁺Feces not available from four cats at time point 26–28 because of early termination of treatment due to severe gastrointestinal signs.

Figure 2. Phylum- and class-level composition of fecal microbiota obtained from feline fecal samples.

Samples were collected at baseline (days 5–7), at the conclusion of antibiotic administration (days 26–28), and after a 6-week washout (days 68–70) from 16 healthy cats, 8 per group,⁺ that received 75 mg clindamycin followed 1 h later by either two capsules of a placebo or synbiotic PO once daily for 21 days. ⁺Feces not available from four cats at time point 26–28 because of early termination of treatment due to severe gastrointestinal signs. Legend for all detected classes is shown, grouped by phylum.

Figure 3. Dual hierarchical dendrogram of metabolites, clustered by pathway, that differed significantly over time in feline fecal samples.

Samples were collected at baseline (days 5–7), at the conclusion of antibiotic administration (days 26–28), and after a 6-week washout (days 68–70) from 16 healthy cats, 8 per group,⁺ that received 75 mg clindamycin followed 1 h later by either two capsules of a placebo or synbiotic PO once daily for 21 days. ⁺Feces not available from four cats at time point 26–28 because of early termination of treatment due to severe gastrointestinal signs.

Figure 4. Principal component analysis (PCA) of metabolic pathway analyses from feline fecal samples.

Samples were collected at baseline (days 5–7), at the conclusion of antibiotic administration (days 26–28), and after a 6-week washout (days 68–70) from 16 healthy cats, 8 per group,⁺ that received 75 mg clindamycin followed 1 h later by either two capsules of a placebo or synbiotic PO once daily for 21 days. ⁺Feces not available from four cats at time point 26–28 because of early termination of treatment due to severe gastrointestinal signs.

Figure 5. Box and whisker plots of fecal metabolite profiles for selected fecal metabolites that differed significantly (fdr - adjusted P < 0.05) between treatment groups and over time. Figure 5a:

(A) Malonic acid. (B) 2-deoxytetronic acid. (C) Threitol. (D) Fumaric acid. (E) N-acetylaspartic acid. (F) Myristic acid. Figure 5b: (A) 5,6-dihydrouracil major. (B) Malic acid. (C) Octadecanol. (D) Palmitoleic acid. Figure 5c: (A) Myo-inositol. (B) 2-hydroxybutanoic acid. (C) Squalene. (D) Dihydrocholesterol. Medians, interquartile ranges, and minimum and maximum values are presented for cats in the placebo (boxes with solid borders) and synbiotic (boxes with dashed borders) treatment groups. Open circles and closed triangles denote outlier values. Fecal samples were collected at baseline (days 5–7), at the conclusion of antibiotic administration (days 26–28), and after a 6-week washout (days 68–70) from 16 healthy cats, 8 per group,⁺ that received 150 mg clindamycin with either a placebo or synbiotic PO once daily for 21 days. ⁺Feces from four cats (placebo group) were unavailable at time point 26–28 because of early termination of treatment due to severe gastrointestinal signs. Significance was set as P < 0.05, with P-values adjusted based on the Benjamini and Hochberg False discovery rate (fdr). Boxes that do not share a letter differed significantly (fdr-adjusted P < 0.05) based on post hoc analysis. For squalene, post hoc analysis did not clarify the group association. Both treatment group and group by time interactions were identified for dihydrocholesterol.

Figure 5b. (Continued).

Figure 5c. (Continued).

Table 2. Metabolites of known biological import with fecal profiles that significantly differed by group by time interaction, group, and/or time.

	Baseline		Days 26-28		Days 68-70
	Placebo	Synbiotic	Placebo^+	Synbiotic	Placebo	Synbiotic	Fdr P-value
Short chain fatty acid metabolites
3,4-dihydroxyphenyl-acetic acid	1,615^a	803^a	425^b	828^b	1,210^a	2,352^a	0.04 (time)
	(962-2,680)	(314-3,602)	(223-724)	(383-2,525)	(710-9,369)	(180-20,565)
3-hydroxyphenyl-acetic acid	529^a	3,514^a	78^c	291^c	214^b	252^b	<0.01 (time)
	(172-4,109)	(289-18,478)	(21-138)	(26-2,221)	(69-12,535)	(152-718)
2,3-dihydroxy-butanoic acid	697^a	175^a	82^b	50^b	146^a	245^a	0.02 (time)
	(222-1,558)	(52-1,606)	(59-306)	(8-226)	(58-1,832)	(111-901)
2-hydroxy-butanoic acid	21,383^a	1,312^b	24,019^a	16,403^b	21,683^a	13,412^b	0.05 (group)
	(2,388-54,254)	(702-19,922)	(4,259-71,903)	(2,007-42,590)	(2,317-49,338)	(1,712-33,500)
3-aminoisobutyric acid	1,567^a	5,230^a	2,696^b	923^b	5,630^a	4,558^a	0.02 (time)
	(832-3,538)	(2,628-6,725)	(89-3,878)	(107-3,548)	(367-7,288)	(929-7,447)
4-aminobutyric acid	4,798^b	2,212^b	40,821^a	5,644^a	17,363^a	12,446^a	0.01 (time)
	(2,330-111,922)	(1,625-14,553)	(18,349-113,143)	(3,811-66,681)	(6,165-28,013)	(1,236-57,235)
2,4-diaminobutyric acid	2,291^a	1,768^a	312^b	323^b	3,413^a	2,981^a	<0.01 (time)
	(1,388-5,136)	(366-9,508)	(167-441)	(172-686)	(266-12,630)	(635-6,901)
4-hydroxybutyric acid	2,269^a	971^a	1,751^a	1,876^a	1,041^b	905^b	0.01 (time)
	(644-8,014)	(452-3,697)	(1,199-3,123)	(1,198-2,240)	(343-1,796)	(273-1,203)
3-phenyllactic-acid	5,318^b	1,366^b	4,709^b	4,363^b	26,433^a	16,257^a	<0.01 (time)
	(523-20,918)	(275-12,731)	(4,529-23,160)	(442-12,880)	(2,799-50,765)	(185-44,885)
Lactic acid	29,969^b	6,457^b	1,046,986^a	288,190^a	473,391^a	188,366^a	0.01 (time)
	(4,279-1,912,722)	(3,129-224,210)	(897,705-1,199,654)	(19,338-1,514,659)	(4,123-1,692,997)	(2,810-1,067,482)
P-hydroxylphenyl-lactic acid	2,775^c	712^c	3,183^b	6,196^b	15,378^a	10,557^a	<0.01 (time)
	(1,076-8,670)	(332-3,808)	(1,372-5,234)	(2,315-16,911)	(3,039-33,947)	(469-42,710)
Propane-1-3-diol	274^b	354^b	4,287^a	3,278^a	1,696^a	1,375^a	<0.01 (time)
	(194-7,900)	(161-653)	(2,020-4,881)	(1,375-6,740)	(495-16,144)	(367-11,753)
3,3-hydroxyphenyl propionic acid	118,316^a	139,062^a	132^c	190^c	1,238^b	948^b	<0.01 (time)
	(55,264-160,927)	(55,351-212,599)	(92-377)	(44-1,634)	(188-190,172)	(170-181,388)
3-(4-hydroxyphenyl)-propionic acid	41,265^a	71,768^a	4,156^c	4,981^c	18,509^b	17,346^b	<0.01 (time)
	(24,170-110,451)	(34,626-143,552)	(1,907-10,735)	(1,442-18,082)	(5,641-48,155)	(4,827-33,522)
Succinic acid	48,717^b	2,100^b	195,371^ab	40,189^ab	218,304^a	299,338^a	0.03 (time)
	(1,935-390,402)	(747-271,563)	(21,751-795,120)	(8,920-1,147,221)	(1,166-674,751)	(2,113-619,092)
Bile acids
Dihydrocholesterol	2,279^b	6,518^ab	1,446^b	5,581^a	3,528^ab	6,203^ab	<0.01 (group by time), <0.01 (group)
	(100-5,528)	(1,182-26,585)	(863-2,691)	(964-7,994)	(281-6,382)	(1,448-12,017)
Deoxycholic acid	78,243^a	38,492^a	299^c	1,358^c	8,383^b	4,594^b	<0.01 (time)
	(838-288,348)	(8,421-922,997)	(81-1,329)	(202-16,475)	(84-298,628)	(35-976,489)
Tryptophan metabolites
Indole-3-lactate	110,344^a	134,566^a	26,768^b	75,418^b	117,419^a	162,374^a	0.01 (time)
	(72,690-224,874)	(33,221-453,636)	(13,271-102,119)	(44,683-184,909)	(65,124-188,639)	(1,430-338,952)
Sphingolipid metabolites
Cellobiose	21,065^a	4,832^a	49,231^a	5,255^a	2,774^b	3,282^b	0.01 (time)
	(2,701-39,188)	(318-37,705)	(8,116-247,769)	(3,566-11,868)	(823-6,438)	(271-8,657)
Isopentadecanoic acid	41,428^a	92,495^a	30,204^b	24,276^b	19,356^b	14,396^b	<0.01 (time)
	(25,355-126,295)	(44,950-173,882)	(26,776-49,252)	(10,430-34,491)	(4,918-256,801)	(4,901-116,494)
Pentadecanoic acid	25,635^a	65,751^a	15,268^b	10,376^b	9,767^b	12,034^b	<0.01 (time)
	(17,767-96,730)	(19,963-118,284)	(5,939-17,510)	(9,721-13,001)	(5,710-13,775)	(6,110-46,327)
Polyamines
Putrescine	592,861^a	400,585^a	165,171^b	82,950^b	192,405^b	221,571^b	0.05 (time)
	(188,643-1,084,643)	(7,787-2,741,972)	(66,399-416,065)	(11,840-672,670)	(16,170-725,961)	(49,993-742,246)
Squalene†	3,289 (1,872-12,906)	6,399 (2,022-18,225)	2,808 (1,806-6,805)	3,397 (1,248-32,188)	5,418 (1,087-11,839)	3,230 (956-15,542)	<0.01 (group)
Antioxidants / antimicrobials
3,4-dihydroxyhydrocinnamic acid	27,522^b	14,896^b	33,604^ab	38,149^ab	40,648^a	148,715^a	0.04 (time)
	(764-39,566)	(1,307-39,007)	(11,036-44,143)	(17,192-78,347)	(1,836-363,292)	(1,501-640,134)
3,4-dihydroxybenzoic acid	10,816^a	4,262^a	2,831^b	3,029^b	7,107^a	19,317^a	0.05 (time)
	(3,732-21,603)	(2,483-20,760)	(2,069-4,290)	(2,092-3,525)	(2,617-33,854)	(1,085-48,873)
4-hydroxybenzoate	22,974^a	11,663^a	4,288^b	4,153^b	16,556^a	19,013^a	0.02 (time)
	(15,351-46,996)	(1,061-24,039)	(1,355-11,641)	(1,726-14,652)	(5,239-44,331)	(1,301-41,178)

Median (range) peak height of metabolites in feces collected at baseline (days 5–7), at the conclusion of antibiotic administration (days 26–28), and after a 6-week washout (days 68–70) from 16 healthy cats, 8 per group,⁺ that received 75 mg clindamycin followed 1 h later by either two capsules of a placebo or synbiotic PO once daily for 21 days.

⁺Feces not available from four cats at time point 26–28 because of early termination of treatment due to severe gastrointestinal signs. Fdr P-value = Benjamini and Hochberg False discovery rate (fdr) adjusted P-value. Profiles that do not share a common superscript letter differed significantly based on post hoc analysis. ^†Post hoc analysis did not clarify source of significant differences between groups.

Table 3. Metabolic pathways that differed significantly over time.

		Total Cmpds	Hits	Fdr P-value	Pathway impact value
1*	Alanine, aspartate and glutamate metabolism	24	10	9.55E-04	0.59
2*	Glycine, serine and threonine metabolism	48	9	5.91E-08	0.54
3°	Galactose metabolism	41	14	1.93E-05	0.52
4*	Arginine and proline metabolism	77	16	1.70E-04	0.48
5*	Taurine and hypotaurine metabolism	20	4	4.27E-04	0.38
6°	Starch and sucrose metabolism	50	8	9.52E-04	0.36
7*	Beta-Alanine metabolism	28	6	6.84E-04	0.34
8	Pyruvate metabolism	32	2	5.02E-03	0.32
9*	Phenylalanine metabolism	45	13	6.49E-07	0.27
10†	Pantothenate and CoA biosynthesis	27	6	8.59E-05	0.25
11*	Cysteine and methionine metabolism	56	7	6.49E-07	0.23
12*	Lysine degradation	47	4	1.86E-04	0.23
13‡	Pyrimidine metabolism	60	9	4.01E-05	0.22
14	Glycerolipid metabolism	32	5	7.93E-06	0.22
15	Histidine metabolism	44	4	1.39E-02	0.21
16	Citrate cycle (TCA cycle)	20	4	1.75E-02	0.21
17°‡	Amino sugar and nucleotide sugar metabolism	88	9	1.17E-02	0.21
18*	Tyrosine metabolism	76	10	4.01E-05	0.20
19	Aminoacyl-tRNA biosynthesis	75	18	9.70E-08	0.17
20†	Butanoate metabolism	40	8	6.19E-03	0.17
21*	Tryptophan metabolism	79	2	2.88E-02	0.16
22‡	Purine metabolism	92	13	5.40E-09	0.12
23*	Phenylalanine, tyrosine and tryptophan biosynthesis	27	5	1.49E-05	0.11
24*	Lysine biosynthesis	32	3	1.11E-04	0.10
25	Glycolysis or Gluconeogenesis	31	4	3.49E-03	0.10
26	Sphingolipid metabolism	25	3	5.32E-03	0.09
27°	Pentose and glucuronate interconversions	53	7	3.81E-02	0.09
28	Glycerophospholipid metabolism	39	2	6.52E-04	0.09
29†	Propanoate metabolism	35	6	2.81E-04	0.09
30*	Valine, leucine and isoleucine biosynthesis	27	6	1.15E-06	0.09
31	Sulfur metabolism	18	3	9.70E-08	0.07
32°	Fructose and mannose metabolism	48	3	2.55E-02	0.07
33	Nitrogen metabolism	39	10	1.47E-04	0.07
34*	Valine, leucine and isoleucine degradation	40	4	7.03E-06	0.06
35	Ubiquinone and other terpenoid-quinone biosynthesis	36	4	4.27E-04	0.05
36	Glyoxylate and dicarboxylate metabolism	50	5	5.68E-03	0.04
37	Caffeine metabolism	21	1	2.37E-04	0.03
38	Fatty acid metabolism	50	2	5.17E-03	0.03
39	Glutathione metabolism	38	5	4.01E-05	0.03
40°	Pentose phosphate pathway	32	4	3.83E-03	0.02
41	Methane metabolism	34	2	8.82E-06	0.02

Pathways that differed significantly over time by group based on fecal metabolite profiles in feces collected at baseline (days 5–7), at the conclusion of antibiotic administration (days 26–28), and after a 6-week washout (days 68–70) from 16 healthy cats, 8 per group,⁺ that received 75 mg clindamycin followed 1 h later by either two capsules of a placebo or synbiotic PO once daily for 21 days.

⁺Feces not available from four cats at time point 26–28 because of early termination of treatment due to severe gastrointestinal signs. Total Cmpds = total compounds in pathway; fdr P-value = Benjamini and Hochberg False discovery rate (fdr) adjusted P-value; * = amino acid metabolism,  = sugar metabolism, † = short-chain fatty acid metabolism, ‡ = nucleotide metabolism.

Figure 6. Study design, duration, observations, and sampling flowchart.

The study spanned 670 days (D1–70) and was broken into three study periods: baseline (D1–D7), treatment (D8–D28), and washout (D29–70). Cats were randomized to receive 75 mg clindamycin PO followed 1 h later by either a placebo or synbiotic once daily during treatment. Food intake, vomiting, and fecal score were recorded daily (•) and weight (W) weekly (*) by an individual blinded to treatment group. From the center portion of each first morning naturally voided fecal sample for each cat, 2 g were collected once daily on the last 3 days of each study period: baseline (open circles), treatment (open squares), and recovery (open diamonds). Each sample was subdivided into two aliquots, with each aliquot placed into a 2 mL cryovial and immediately frozen at –80ºC pending completion of data collection.