699 - Interplay Between Endogenous Glycan Fermentation and Klebsiella Pathogenicity in the Intestinal Lumen

Publication Number: 699.5138

Surabhi Khasgiwala, Connecticut Children's Medical Center, Farmington, CT, United States; Katrin Unterhauser, University of Connecticut School of Medicine, Farmington, CT, United States; Karim Rezaul, University of Connecticut School of Medicine, Farmington, CT, United States; Sonam Tamrakar, University of Connecticut, Storrs Mansfield, CT, United States; jeremy Balsbaugh, University of Connecticut, Storrs, CT, United States; Anthony A. Provatas, University of Connecticut, Storrs, CT, United States; adam matson, Connecticut Children's Medical Center, Hartford, CT, United States

Background: Cytotoxin (tilimycin)-producing members of Klebsiella oxytoca species complex (KoSC) are the causative agent of antibiotic-associated hemorrhagic colitis (AAHC) and have been linked to necrotizing enterocolitis (NEC) in premature infants; however, they are also found in the gut of subjects that do not develop intestinal disease. The contextual basis for KoSC to produce tilimycin and transition from commensal to pathogen remains poorly understood.

Objective: To determine if endogenous glycans induce tilimycin synthesis and define host exposures that increase luminal glycans for KoSC consumption.

Design/Methods: K. oxytoca AAHC isolate (AHC-6) and K. grimontii NEC isolate (UCH-1) were cultured in lysogeny broth (LB) +/- glucose, fucose, or sialic acid. Growth was measured by optical density at 600 nm (OD600). Cytotoxin production was assessed using bacterial culture supernatants applied to T84 intestinal epithelial cells and via mass spectrometry (MS). UCH-1 ∆fucI and ∆nanT mutants were generated, which lack genes required for fucose and sialic acid metabolism, and growth and cytotoxin production were similarly assessed. C57BL6/J mice were treated or not with amoxicillin and/or indomethacin, and levels of free fucose and sialic acid were quantified in cecal contents by liquid chromatography MS. Kruskal-Wallis tests with Dunn’s multiple comparisons assessed differences between groups.

Results: All three sugars enhanced tilimycin production and cytotoxicity compared to when the isolates were cultured in LB alone. Fucose and sialic acid supported KoSC growth and induced tilimycin production to an equivalent or greater level as glucose. UCH-1 ∆fucI and ∆nanT mutants failed to make tilimycin in LB + fucose or sialic acid respectively. Free sialic acid increased in murine cecal contents 24 – 72 hours following the initiation of amoxicillin and/or indomethacin treatment (Figure), whereas fucose remained below the limit of detection.

Conclusion(s): Sialic acid and fucose induce in vitro tilimycin production by KoSC. Treatment with antibiotics and/or indomethacin increases luminal sialic acid availability potentially driving KoSC pathogenicity in vivo. Future studies will assess luminal tilimycin production and intestinal mucosal damage in mice inoculated with UCH-1 wild-type vs. ∆nanT and treated with amoxicillin +/- indomethacin.

Figure.
Concentrations of free sialic acid (Neu5Ac) in murine cecal contents at 24- and 72-hours following treatment with amoxicillin (100 mg/kg) twice daily by intraperitoneal injection and/or indomethacin (2.5 mg/kg) once daily by subcutaneous injection. *P ≤ 0.05; **P ≤ 0.01.

Figure.
Concentrations of free sialic acid (Neu5Ac) in murine cecal contents at 24- and 72-hours following treatment with amoxicillin (100 mg/kg) twice daily by intraperitoneal injection and/or indomethacin (2.5 mg/kg) once daily by subcutaneous injection. *P ≤ 0.05; **P ≤ 0.01.