K. A. Schall1, J. W. Debelius3, K. A. Holoyda1, R. Knight3, T. C. Grikscheit1,2 2Keck School Of Medicine,Los Angeles, CA, USA 3University Of Colorado Boulder,Department Of Chemistry And Biochemistry,Boulder, CO, USA 1Children’s Hospital Los Angeles,Department Of Pediatric Surgery,Los Angeles, CA, USA
Introduction:
Short bowel syndrome (SBS) is characterized by malnutrition, mucosal inflammation and small intestine bacterial overgrowth (SIBO). The definition and evaluation of SIBO is poorly understood but hypothesized to arise from stasis of luminal contents and colonization from the colon. In some cases, prophylactic antibiotics are given. Previous studies characterized only fecal and colonic mucosal samples, but the small intestinal milieu is known to be quite different. We recently developed a novel model of SBS in the zebrafish: a high proximal stoma is formed at the fish correlate to the human jejunum, with a distal Hartman’s pouch. As in the human disease, weight loss and intestinal dilation are noted. We aimed to characterize the small intestine microbiome alterations associated with SBS in our novel zebrafish SBS model.
Methods:
The SBS surgery consisted of a ventral laparotomy, proximal stoma creation, distal bowel ligation and removal of middle portion of intestine to prevent fistula formation. Nineteen adult male zebrafish underwent SBS surgery with proximal ostomy and distal ligation, sham with ventral laparotomy only (n=9), or control with no operation (n=10); half housed separately and together to control for environment. The mid-portion of the intestine was removed at the time of SBS operation while the proximal and distal intestine were harvested from the remaining fish after 2 weeks. DNA was extracted with the PowerSoil kit and 16s rRNA based gene sequencing completed to identify the bacterial species present. Alpha rarefaction to 2500 segments/sample was used to balance loss of samples. Unweighted Unifrac distance was compared using permanova and anosim test. Alpha diversity was compared by kruskal wallis. Paired samples within an individual zebrafish, which consisted of the proximal (“fed”) and distal (“unfed”) intestine, were compared using kruskal wallis. Qiime was used to generate taxonomy, which was plotted using Excel.
Results:
Surgery increased the operational taxonomic units (OTUs) from the genus Shewanella and decreased OTUs from the family Aeormonadacaea and genus Clocibacterium after SBS surgery as compared to the controls. Environment consisting of the tank water was significantly different than the intestinal bacteria (p=.001), having no effect on intestinal microbiota changes. The paired controls of proximal and distal intestine in SBS, despite relative changes in some OTUs, revealed a core microbiota that is unchanged (p<0.01).
Conclusion:
The microbiota is altered in the small intestine after SBS in a zebrafish model, possibly correlating to the human disease process, leading to increased inflammation and SIBO. Understanding the luminal factors after surgery, including the microbiome, may indicate novel therapeutic interventions for children with this morbid disease.