Published online Apr 14, 2021. doi: 10.3748/wjg.v27.i14.1465
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
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.
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.
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).
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.
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.
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.
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.