Retrospective Cohort Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 14, 2021; 27(14): 1465-1482
Published online Apr 14, 2021. doi: 10.3748/wjg.v27.i14.1465
Impact of preoperative antibiotics and other variables on integrated microbiome-host transcriptomic data generated from colorectal cancer resections
Sarah A Malik, Chencan Zhu, Jinyu Li, Joseph F LaComb, Paula I Denoya, Igor Kravets, Joshua D Miller, Jie Yang, Melissa Kramer, W Richard McCombie, Charles E Robertson, Daniel N Frank, Ellen Li
Sarah A Malik, Joseph F LaComb, Igor Kravets, Joshua D Miller, Ellen Li, Department of Medicine, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, United States
Chencan Zhu, Jie Yang, Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794, United States
Jinyu Li, Jie Yang, Stony Brook Cancer Center Biostatistics and Bioinformatics Shared Resource, Stony Brook University, Stony Brook, NY 11794, United States
Jinyu Li, Department of Pathology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, United States
Paula I Denoya, Department of Surgery, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, United States
Jie Yang, Department of Family, Population and Preventive Medicine, Stony Brook University, Stony Brook, NY 11794, United States
Melissa Kramer, W Richard McCombie, Cold Spring Harbor Laboratory Cancer Center Sequencing Technologies and Analysis Shared Resource, Cold Spring Harbor, NY 11724, United States
Charles E Robertson, Daniel N Frank, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States
Author contributions: Li E, McCombie WR, Denoya PI, Frank DN, Kravets I and Miller JD contributed study concept and design; Malik SA, LaComb JF, Li E, Kramer M contributed data collection; Zhu C, Yang J, Li J, Robertson CE and Frank DN contributed statistical analysis; Malik SA, Li E and Frank DN contributed drafting of manuscript; Yang J, LaComb JF, Denoya PI, Kravets I, Miller JD, Kramer M, McCombie WR and Robertson CE contributed critical review for important intellectual content; all authors approved the final version of the manuscript.
Supported by National Cancer Institute, No. P20 CA192994; National Cancer Institute, No. P20 CA192996; and Simons Foundation, No. 415604.
Institutional review board statement: This study was approved by the Stony Brook University Institutional Review Board (approval No. 163184).
Informed consent statement: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.
Conflict-of-interest statement: All authors declare no conflicts-of-interest related to this article.
Data sharing statement: All de-multiplexed, paired-end 16S rRNA gene sequence files and RNA-sequence data along with associated metadata were deposited into the Gene Expression Omnibus, which is a public repository that archives and freely distributes comprehensive sets of microarray, next-generation sequencing, and other forms of high-throughput functional genomic data submitted by the scientific community data base under project accession number GSE165255. All the tissues analyzed were collected from patients that gave informed consent for sharing results of the analysis of their anonymized tissues on public databases, where the risk of identification is very low. The statistical code used in this study is available from the corresponding author at
STROBE statement: The authors have read the STROBE Statement-checklist of items, and the manuscript was prepared and revised according to the STROBE Statement-checklist of items.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Ellen Li, MD, PhD, Emeritus Professor, Department of Medicine, Renaissance School of Medicine, Stony Brook University, 101 Nicolls Road, Stony Brook, NY 11794, United States.
Received: January 21, 2021
Peer-review started: January 21, 2021
First decision: February 9, 2021
Revised: March 3, 2021
Accepted: March 24, 2021
Article in press: March 24, 2021
Published online: April 14, 2021
Research background

Imbalances in mucosal associated microbiota (dysbiosis) have been reported in human colorectal cancer (CRC). There are certain pathogens that are associated with CRC including increased abundances of Peptostreptococcus, Bacteroides fragilis, Fusobacterium nucleatum, and Escherichia coli and decreased abundances of Clostridium, Bifidobacterium, Faecalibacterium and Roseburia. The approach of collecting and integrating multi-omic host and microbiome data has been increasingly applied to discovery and functional studies of human gastrointestinal disease.

Research motivation

A major change was made in the preoperative antibiotic protocol at this hospital as of January 2017. Prior to that time, the standard of care was to administer only intravenous antibiotics within 30 min of incision, and only a few CRC resection patients were placed on short-term oral antibiotics within a month of the surgery, for various clinical indications. However, after this time, the standard operating protocol for preoperative antibiotics was to prescribe outpatient oral neomycin and metronidazole 24 h in advance of the procedure, in order to reduce surgical site infections. The use of antibiotics can shift the microbiome depending on the dosage and duration of the antibiotic exposure. Several studies have shown that tumorigenesis and tumor growth can be attenuated with different antibiotic cocktails and timing of antibiotic exposure with duration of inflammation. On the other hand, early exposure to antibiotics increased risk of CRC and interfered with chemotherapy efficacies due to microbial dysbiosis. With these conflicting findings and this change in protocol at our institution, it allowed us to examine how differential use of antibiotics, along with other clinical/demographic factors influences integrative, multi-omic analyses of CRC.

Research objectives

To examine the effect of the five following variables were included in the analysis of the microbiome and host transcriptome datasets generated in this pilot study: (1) Tumor histology (tumor vs nontumor); (2) Preoperative antibiotics (yes/no); (3) Laterality of CRC location (left vs right); (4) Diabetes mellitus (yes/no); and (5) Black/African Ancestry (AA) race (yes/no).

Research methods

Genomic DNA (gDNA) and RNA were extracted from prospectively collected 51 pairs of frozen sporadic CRC tumor and adjacent non-tumor mucosal samples from 50 CRC patients archived at a single medical center from 2010-2020. 16S rRNA gene sequencing (V3V4 region, paired end, 300 bp) and confirmatory quantitative polymerase chain reaction (PCR) assays were conducted on gDNA. RNA sequencing (IPE, 125 bp) was performed on parallel tumor and non-tumor RNA samples with RNA Integrity Numbers scores ≥ 6.

Research results

Exploratory PERMANOVA using the Morasita Horn index for β-diversity was performed with each of the five groups. Tumor vs nontumor histology (P = 0.002) and preoperative antibiotics (P = 0.001) demonstrated significant F statistics, but anatomic location, diabetes and Black/AA race did not reach significance. Differences in α-diversity did not reach significance between the five groups. Fourteen taxa at the genus level exhibited significant tumor*preoperative antibiotic interactions. Of the taxa without tumor*preoperative antibiotic interactions, 7 taxa were significantly increased in tumor vs nontumor, including Fusobacterium, and 11 taxa were significantly decreased. The increase in Fusobacterium nucleatum (F. nucleatum) abundance was confirmed by Taqman PCR assays. Additional preoperative antibiotics significantly reduced mucosa-associated total bacterial abundance, which may contribute to reduction of intra-abdominal surgical site infections. Analysis of a subset of parallel formalin-fixed paraffin embedded (FFPE) samples retained polarity of the observed trends but impaired signal strength. Principal coordinate analysis of the transcriptomic data showed a clear separation from tumor and nontumor samples. Consequently, differentially expressed genes were analyzed separately for the other four variables separately in tumor and nontumor samples. Differentially expressed genes common to the tumor and nontumor groups were identified for additional pre-operative antibiotics and Black/AA race. The VBP1 gene, which may suppress CRC metastasis, exhibits reduced expression in Black/AA subjects compared to not Black/AA subjects.

Research conclusions

The recent addition of preoperative oral antibiotics 24 h to the standard administration of IV antibiotics within 30 min of incision has a measurable effect on colonic mucosal gene expression in addition to its effect on the amount and composition of mucosal associated bacteria in the resected specimen. Despite heterogeneity in the preoperative antibiotics in this study cohort, increased abundance of F. nucleatum, was observed in tumor vs nontumor regions of the resected specimen. This study identified the VBP1 gene, which may suppress CRC metastasis, as having decreased RNA expression in both tumor and nontumor regions of the resected specimen collected from Black/AA subjects.

Research perspectives

Given that the addition of preoperative antibiotics to the standard administration of IV antibiotics within 30 min of incision has a measurable effect on colonic mucosal gene expression, in addition to its effect on the amount and composition of mucosa-associated bacteria in the resected specimen, is being widely adopted as standard of care, we plan to perform targeted PCR assays on archived CRC FFPE tissues to confirm our results collected prior to when the change in protocol was adopted (January 2017). Because patients undergoing colonoscopy are not routinely prescribed antibiotics before the procedure, we also plan to shift prospective collection of colonic neoplastic and normal tissues away from surgical resections to prospective collection of research colonoscopic biopsy samples.