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Page 6 of 12 Horie et al. Microbiome Res Rep 2024;3:35 https://dx.doi.org/10.20517/mrr.2024.08

isolated from horse feces. The herbivore samples also contained Weissella confusa and Streptococcus sp. No
former Lactobacillus species were isolated from giraffe feces. Ligilactobacillus animalis L. animalis was
isolated from the feces of Asian black bear and sun bear, which tend to be herbivorous. By contrast,
Ligilactobacillus agilis and L. saerimneri were isolated from the feces of polar bears. Although the western
Indian manatee is an herbivore, no former Lactobacillus species were isolated from its feces; instead, the
colonies that formed on the MRS agar plates were Lactococcus garviae and E. faecalis. Similarly, no former
Lactobacillus species were isolated from the piscivores; instead, Lactococcus garvieae (L. garvieae),
Streptococcus sp., and Enterococcus sp. were isolated as lactic acid bacteria from these animals.

Sugar utilization of Lactobacillaceae isolated from the carnivores and herbivores
Sugar utilization by Lactobacillaceae isolated from the carnivores and herbivores was examined
[Supplementary Table 3]. Sugar utilization was more dependent on the bacterial species than on the source
of isolation. D-Glucose, D-fructose, N-acetyl glucosamine, and D-sucrose were available in all isolated
strains. The types of sugars available to L. saerimneri tended to be fewer than those of other bacterial
species. Meanwhile, significantly more types of sugars were available for the two strains of L. saerimneri,
TOB0030 (JCM36439) and TOB1106 (JCM36449). Twenty-three and 19 sugars were available to the
L. saerimneri strains isolated from lion and polar bear samples, respectively. Galactose was unavailable to
most L. saerimneri strains, but was available to other species. Furthermore, lactose, melibiose, mannitol and
raffinose could not be used in L. saerimneri, but many other species could use them. On the other hand,
trehalose, which was not available in many strains, was available in all strains of L. saerimneri. Among the
L. salivarius strains isolated from carnivores and herbivores, those isolated from the family Felidae could use
10-14 of the 49 types of sugars examined. By contrast, L. salivarius strains isolated from the horse samples
used only 16 types of sugar. In particular, L. salivarius strains isolated from horse samples were able to use
xylitol and D-arabitol.

Principal coordinate analysis and cluster analysis
Since the types of lactic acid bacteria tended to differ depending on the feeding habit rather than the species
of the host, the intestinal flora was analyzed next. To compare the diversity of the fecal bacterial flora among
the animals, UniFrac distance was calculated, and PCoA was performed [Figure 1]. According to
unweighted UniFrac analysis, the fecal flora of carnivorous animals showed similarities. The fecal flora of
herbivores also showed similarities regardless of whether they were terrestrial, such as the horse, or aquatic,
such as the manatee. According to weighted UniFrac analysis, the fecal flora of piscivores showed
similarities, in addition to those of carnivores and herbivores. These results suggest that the bacterial
components and their proportions in fecal flora depend on the feeding habits of the host animals.

Fecal floras of the carnivores, piscivores, herbivores, and omnivores
Fecal floras of the carnivores, piscivores, herbivores, and omnivores were analyzed
[Supplementary Table 4]. Differences in the composition of fecal flora were observed at the phylum level
based on feeding habits. Despite Bacillota being the most dominant regardless of feeding habits, herbivores
exhibited a higher proportion of Bacteroidota. The average percentages of Bacteroidota were 7.7%, 58.5%,
and 46.2% for carnivores, herbivores, and piscivores, respectively. Conversely, among piscivores, there was a
higher proportion of Proteobacteria, followed by Fusobacteriota. At the order level, carnivores showed
relatively high proportions of Peptostreptococcales-Tissierellales. In herbivores, Bacteroidales predominated,
while Enterobacterales and Fusobacteriales were more prevalent in piscivores. Additionally, carnivores
exhibited a high proportion of Peptostreptococcaceae at the family level, along with notable proportions of
Coriobacteriaceae, Clostridiaceae, and Fusobacteriaceae. Herbivores displayed a relatively dispersed
distribution of bacterial proportions at each level. Similarly, piscivorous animals also showed dispersed
proportions, with Peptostreptococcaceae, Oscillospiraceae, and Fusobacteriaceae being relatively dominant.