IDENTIFYING THE ROLE OF ACINETOBACTER CALCOACETICUS IN DRIVING INTESTINAL INFLAMMATION

Abstract

Abstract BACKGROUND Inflammatory bowel disease (IBD) is a life-long condition characterized by chronic inflammation of the gastrointestinal tract. Among the potential causes of IBD, the gut microbiota has been implicated in perpetuating inflammation. Analysis of the IBD Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) database revealed that Acinetobacter calcoaceticus is one of the top 10 highest elevated bacteria in Crohn’s’ disease patients. Acinetobacter are well-characterized for their antibiotic resistance, but little is known about their interaction with the gut. We hypothesize that A. calcoaceticus colonizes the gut and promotes intestinal inflammation. MATERIALS & RESULTS We grew commercially available and clinical isolates of A. calcoaceticus in vitro with stressors found in the gut. All strains grew in a range of pHs (4-7), osmolarity (0.1-1 M NaCl), ethanol (1- 5%) and hydrogen peroxide (0.05-0.1%); indicating that A. calcoaceticus is well-adapted to withstand the harsher conditions of the gastrointestinal tract. To further investigate the ability of A. calcoaceticus to colonize the gut, we utilized Caenorhabditis elegans as a model. All A. calcoaceticus strains colonized the nematode's gut. We likewise found that all A. calcoaceticus strains colonized human stool-based bioreactors; suggesting that A. calcoaceticus can colonize the gastrointestinal tract. To identify whether A. calocaceticus stimulated inflammation, we incubated intestinal organoids and macrophages with live A. calcoaceticus or LPS derived from our A. calcaoceticus strains and found that both conditions stimulated pro-inflammatory cytokines. In order to confirm our in vitro findings, we oral gavaged adult BalbC mice with A. calcoaceticus and examined fecal levels using selective agar and qPCR. Acinetobacter was observed at low levels in the vehicle control mice and at high levels in our mice gavaged with A. calcoaceticus (average 9.9x 107 CFU); suggesting that A. calcoaceticus colonized the murine gut. To examine the ability of A. calcoaceticus to enhance inflammation, we oral gavaged mice with either a vehicle control (PBS) or A. calcoaceticus and the following day we administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) to induce colitis. Mice treated with A. calcoaceticus and TNBS lost more weight and had worse histological scores than mice treated with vehicle control and TNBS; indicating A. calcoaceticus worsens intestinal inflammation. Finally, to expand the niche of Acinetobacter, we pre-treated mice with antibiotics and then subsequently administered bacteria and TNBS. In this model, A. calcoaceticus exacerbated inflammation above that of non-antibiotic treated mice. CONCLUSIONS These data show that A. calcoaceticus can promote intestinal inflammation and may be a contributing factor to the inflammation experienced by IBD patients.