723 - Microbiota Profiles in Surgical Necrotizing Enterocolitis: Insights from FFPE Tissue Analysis Reveal Dysbiosis in Bacterial Communities in Non-Survivors

Publication Number: 723.5621

Parvesh Mohan Garg, Wake Forest School of Medicine of Wake Forest Baptist Medical Center, winston salem, NC, United States; Pengbo Zhang, Wake Forest School of Medicine of Wake Forest Baptist Medical Center, Winston-Salem, NC, United States; Greg A. Hawkins, Wake Forest School of Medcine, Winston Salem, NC, United States; Kylie Kavanagh, Wake Forest School of Medicine of Wake Forest Baptist Medical Center, Winston Salem, NC, United States; Liang Liu, wake forest university school of medicine, winston salem, NC, United States; Timothy Howard, Wake Forest School of Medicine of Wake Forest Baptist Medical Center, Winston-Salem, NC, United States; Avinash K. Shetty, Wake Forest University School of Medicine, Winston-Salem, NC, United States; Wei Zhang, Wake Forest School of Medicine of Wake Forest Baptist Medical Center, Winston Salem, NC, United States; Padma Garg, University of Mississippi Medical Center, Jackson, MS, United States

Background: Necrotizing enterocolitis (NEC) is a common cause of death in preterm infants and is associated with changes in gut microbiome. Existing studies are largely limited to non-invasive stool samples, which may not be reflective of the bacteria located at anatomical sites of disease.

Objective: We aimed to understand the microbiota profiles in survivors and non-survivors in preterm infants with surgical NEC by analyzing historical formalin fixed paraffin-embedded (FFPE) intestinal tissue.

Design/Methods: We analyzed FFPE small intestine tissues from 16 NEC infants (8 survivors and 8 non survivors). Extracted DNA from FFPE tissue blocks underwent 16S rRNA sequencing targeting the V3-V4 region. The trimming, denoising, and de-multiplexing were performed using DADA2.For taxonomy assignment, a Naïve-Bayes classifier was trained using the latest SILVA SSU database (version 138.1) as the reference. Reads classified as Archaea, along with those that could not be assigned at the phylum level, were discarded.
Statistical Analysis: The abundance levels of different microbiota in each sample were directly compared using the Mann-Whitney U rank sum test, with p-values below 0.05 deemed significant.For each individual, Shannon entropy, and other metrics were calculated as Alpha diversity metrics. For Beta diversity, Unweighted and weighted UniFrac distances was used to calculate the distance between all possible sample pairs. The weighted UniFrac distances between sample pairs were visualized using UMAP, revealing a distinct community structure between the Survivor and Non-survivor groups.

Results: Sequencing of FFPE extracted DNA resulted in high quality sequence reads in all cases. Beta diversity on UMAP showed survivor and non-survivor group formed different communities. Based on Shannon’s entropy, the non-survivor group had a more complex microbiota composition than survivors (p=0.02) that exhibited higher Pielou evenness than survivors (p=0.017). The survivor group had higher counts of observed features than non survivors (p=0.004). indicating high beta diversity. The clinical groups based on necrosis, inflammation, and hemorrhage did not show distinct communities based on alpha and beta diversity. Histopathological features of NEC severity did not relate to microbial community profiles unlike survival outcome. The data are summarized in Figure 1, Figure2 and Figure 3.

Conclusion(s): The Intestinal FFPE blocks provide a robust and specific material for profiling the microbiota at the disease site. The non-survivors had complex microbiota and had a distinct bacterial communities compared to survivors.

Fig1. Alpha diversity comparison between groups, and the corresponding p-values
Fig1. Alpha diversity comparison between groups, and the corresponding p-values

Fig 2. Class level bacteria abundance between two groups with p<0.05
Fig 2. Class level bacteria abundance between two groups with p<0.05

Figure 3: Community structure of survivor groups with respect to beta diversity metrics
Figure 3: Community structure of survivor groups with respect to beta diversity metrics

Fig1. Alpha diversity comparison between groups, and the corresponding p-values
Fig1. Alpha diversity comparison between groups, and the corresponding p-values

Fig 2. Class level bacteria abundance between two groups with p<0.05
Fig 2. Class level bacteria abundance between two groups with p<0.05

Figure 3: Community structure of survivor groups with respect to beta diversity metrics
Figure 3: Community structure of survivor groups with respect to beta diversity metrics