Study 2: How does sampling methodology (swab vs feces) affect
recovered community composition?
Shannon diversity index values were
significantly higher in fecal pellets than in pre-defecation cloacal
swabs (t = 16.29, df = 7, p < 0.001; Fig 4a), as was richness
(t = 8.39, df =,7, p < 0.001; Fig 4b) and phylogenetic
diversity (F = 10.94, df = 7, p < 0.001; Fig 4c). Beta
diversity analysis using Bray-Curtis distance showed that the microbial
composition of fecal pellets was significantly more similar to other
fecal pellets than it was to that of the pre-defecation cloacal swabs (F
= 9.65, df = 1,14, p = 0.002; Fig 5a). The groups were also dispersed
differently (F = 5.54, df = 1,14, p = 0.034). Fecal pellets also
differed from pre-defecation cloacal swabs in both UniFrac metrics
(weighted: F = 18.91, df = 1,14, p = 0.001, Fig 5b; unweighted: F =
15.13, df = 1,14, p = 0.001, Fig 5c), and dispersion of the communities
also varied for each measurement (weighted: F = 13.83, df = 1,14, p =
0.002; unweighted: F = 4.78, df = 1,14, p = 0.046).
There was no significant
difference between pre- and post-defecation swabs in any alpha diversity
metric (Shannon: t = 1.40, df = 7, p = 0.204; Richness: t = 1.45, df =
7, p = 0.190; Phylogenetic diversity: t = 1.72, df =,7, p = 0.129; Fig
4), although the post-defecation swab tended to have higher diversity.
Swab types did not cluster separately based on Bray-Curtis distances (F
= 1.24, df = 1,14, p = 0.231, Fig 5a), and the groups were dispersed
similarly (F = 2.57, df =1,14, p = 0.131). Pre-defecation swab
communities differed from post-defecation swab communities in dispersion
based on weighted (F = 7.08, df = 1,14, p = 0.019, Fig 5b) and
unweighted (F = 36.14, df = 1,14, p < 0.001, Fig 5c) UniFrac
distances, as well as composition of unweighted UniFrac distance (F =
2.20, df = 1,14, p = 0.035), although this could be due to differences
in dispersion rather than true clustering of groups. The different swab
types did not cluster separately based on weighted UniFrac distance (F =
1.14, df = 1,14, p = 0.281).
The most abundant taxa recovered
from fecal pellets was Lachnospiraceae, which made up 38.7 ±
5.2% on average (Fig 6). Enterobacteriaceae, Bacteroidaceae, andRuminococcacea each accounted for ~10% of the
recovered communities, and several others made up between 1-5% of the
recovered community including Tannerellaceae, Erysipelotrichaceae,
Eggerthellaceae, Marinifilaceae, and Akkermansiaceae.Pre-defecation cloacal swab communities were dominated byEnterobacteriaceae (84.9 ± 5.3%), and the next most abundant
taxa was Helicobacteraceae, which only accounted for 5.1 ± 2.2%
of the community. A corncob analysis confirmed thatLachnospiraceae was significantly more abundant (t = -7.20, p
< 0.001) and Enterobacteriaceae was significantly less
abundant (t = 3.83, p < 0.001) in fecal samples compared to
pre-defecation cloacal swabs.
While the same two taxa dominated
the pre- and post-defecation swabs, post-defecationEnterobacteriaceae was reduced to only 44.8 ± 13.2%, andHelicobacteraceae increased to 19.0 ± 9.5%. There was also an
increase in the abundance of feces-associated taxa, particularlyLachnospiraceae which accounted for 15 ± 7.4% on average.
However, there was a distinct bifurcation in the post-defecation swab
samples. In five samples, Enterobacteriaceae andHelicobacteraceae account for >75% of the
community, similar to the pre-defecation swabs, while the remaining
three samples had a more “feces-like” community, with a higher
abundance of Lachnospiraceae and Bacteroidaceae .