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Page 6 of 16 Tejiram et al. Plast Aesthet Res. 2025;12:9 https://dx.doi.org/10.20517/2347-9264.2024.109

Diversity and differential abundance analysis
Alpha diversity was calculated by subsampling the ASV table at 10 different depths (100 to 1,000 sequences)
for Faith’s Phylogenetic Diversity, Observed Features, and Pielou’s Evenness metrics [35,36] . At every depth,
twenty iterations were performed, with their results being averaged for each of the different metrics. A
rarefaction plot was created to confirm that diversity approached an asymptote and slope decreased as
sampling depth increased. Averages for the greatest depth (1,000 sequences) were used to determine if any
of the alpha diversity metrics differed significantly based on the antibiotics group (Yes/No) within the same
time point (Intraop, Dressing, and Follow-Up) (Kruskal-Wallis, P ≤ 0.05). Kruskal-Wallis tests were also
performed to evaluate overall alpha diversity among all groups within the same sample type.

Beta diversity analysis was conducted with the ASV table, which was first normalized with the cumulative
sum scaling method . Distances between samples were calculated using the weighted Unifrac metric based
[37]
on the normalized table and rooted tree and visualized as a Principal Coordinates Analysis (PCoA) plot for
[38]
both buccal swab and wound bed swab samples . Statistical differences within time points based on
antibiotics were evaluated, as well as overall differences among all groups within the same sample type
(PERMANOVA, P ≤ 0.05).

LEfSe was used to identify taxa that had significantly different abundances based on the administration of
[39]
antibiotics within the buccal swab and wound bed swab time points . The ASV table was collapsed to the
species level and normalized with the counts per million method. Enriched taxa were those taxa that were
identified as having significantly differential abundance (Kruskal-Wallis, P ≤ 0.05) with a log(LDA) score of
at least 2.0.

Assessing graft loss and graft-loss grading scale
Digital pictures were used to assess reepithelization in autografted and xenografted wounds by three
independent, blinded burn surgery attendings at patient follow-up visits. The percentage of re-
epithelialization was assessed from zero to 100% and the grade of graft loss was determined. Graft loss
[40]
grading was determined based on a previously published scale, as summarized in Table 1 . The percentage
of re-epithelialization and graft loss grade were then collated from assessors and summarized for
comparison purposes.

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 9.0. with P ≤ 0.05 deemed as statistically
significant. Student’s t-tests were used to compare percent reepithelization between ABX or non-ABX
autografted and xenografted wounds. Results are reported as standard error of the mean (SEM). Univariate
analysis was used to compare relationships in demographic data. Culture data were assessed for normality
using Anderson-Darling normality tests and all data were nonparametric. Un-paired Mann-Whitney tests
were used to assess culture data and nonparametric data are presented as median and interquartile range.

RESULTS
Patient demographics
Of the patients assessed for eligibility, 31 patients were prospectively studied [Figure 1]. The average time
from admission to OR was 2.2 days (IQR, 1-3). Patients had a mean age of 40.8 ± 14.2, with the majority
being male (64.5%). The mean TBSA was 3.4 ± 2.8. Of the study participants, 13 received antibiotics (41.9%)
and 18 received center-specific standard of care (58.1%). Of the 31 patients, 18 patients received autografts
and 13 received xenografts. Among the ABX group, about half were autografted (n = 7, 53.9%) and the other
half were xenografted (n = 6, 46.2%). In the no-ABX group, 11 (61.1%) patients received autografts and
seven (38.9%) received xenografts. Between the ABX and no-ABX groups, there was no difference in length