Background: Hyperammonemia associated with chronic liver disease (cirrhosis) plays a major role in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) that contributes to systemic hyperammonemia in HE. Probiotic bacteria have shown benefits in the treatment of HE although the underlying mechanism(s) are not completely understood. We engineered Escherichia coli Nissle 1917 bacterium (EcN) to consume NH3 and convert it to arginine in the gut. To enhance the beneficial effect of NH3 consumption, we further engineered the EcN to synthesize the short chain fatty acid butyrate in the gut. The resulting strains, SYNARG (arginine producing) and SYNARG+BUT (arginine and butyrate producing) were tested in two experimental models of cirrhosis and HE: thioacetamide (TAA) or bile duct ligation (BDL). Methods: Cirrhosis was induced in BALB/c mice by intraperitoneal treatment with TAA for 4 weeks, and in Sprague-Dawley rats by BDL for 5 weeks. TAA-treated mice were gavaged with a daily dose of 1 x 1010 colony forming units (CFU) of SYNARG. BDL rats were gavaged with 1 x 1012 CFU with SYNARG or SYNARG+BUT for 4 weeks. Plasma NH3 was measured in both models at baseline (BL) and after treatment, with an additional measurement at 2 weeks post treatment (3 weeks post-BDL). Results: TAA mice developed hyperammonemia (BL: 22.4 ± 3.3 umol/L to 4 weeks: 82.4 ± 8.7 umol/L, p<0.05) which was attenuated after SYNARG treatment (45.5 ± 4.7 umol/L, p<0.05). Longitudinal analysis in Vehicle-BDL rats developed hyperammonemia at 3 weeks (BL: 68.8 ± 5.7 to 121.6 ± 9.8 umol/L, p<0.01) which was further increased after 5 weeks (158.8 ± 22.0 umol/L, p<0.001 vs BL and 3 weeks). At 3 weeks, SYNARG+BUT prevented a significant increase in blood NH3 in BDL rats (99.6 ± 8.7 umol/L, p<0.05 vs BL). Both SYNARG+BUT (115.9 ± 17.2 umol/L) and SYNARG (127.9 ± 15.5 umol/L) were protective in preventing further increase in blood NH3 from 3 to 5 weeks, as observed in Vehicle-BDL rats. Moreover, the analysis between groups at 5 weeks showed that SYNARG+BUT reduces NH3 compared to Vehicle-BDL rats (p<0.05). Conclusion: Our data suggest that EcN, engineered to consume NH3 in the gut, is an effective approach to lower plasma NH3 in models of cirrhosis and hyperammonemia. Based on these results, the therapeutic potential of these engineered EcN strains should be further evaluated in patients with liver disease and HE.
