OBJECTIVE: Bacterial microcolonies in gallstones provide a reservoir for biliary infections. Bacterial slime production facilitates adherence, while β-glucuronidase(bG) and phospholipase(PhL) generate colonization surface. These factors are known to facilitate infectious gallstone formation. But, to cause severe infections (and bacteremia) these bacteria need to detach from the biofilm and reflux into the systemic circulation (cholangiovenous reflux). We previously reported that abundant slime inhibited cholangiovenous reflux in rats. The influence of these bacterial factors (slime, bG/PhLprod) on the development of severe infections and bacteremia in a clinical setting has not been studied. In this study we examined the influence these factors on infection severity and bacteremia. METHODS: 260 patients with gallstone disease were studied. Gallstones, bile, and blood (as applicable) were cultured. Bacteria were tested for bG/PhLprod and quantitative slime production. Severe infections (bacteremia, cholangitis, abscess, hypotension, organ failure) were correlated with the bacterial factors (slime, bG/PhLprod). RESULTS: Biliary bacteria were present in 45% of patients, 17% with bacteremia. Severe infections varied directly with bG/PhLprod (50% with vs 17% without, P=0.004) and inversely with magnitude of slime production (52% vs 8%, slime <75 or >75, P=0.004). Low (<75) slime production and bG/PhLprod were additive: severe infections were present in 60% with both, but 11%-16% with either/none (P<0.03). bG/PhLprod facilitated bactibilia (84% with vs 50% without, P=0.003), while slime production trended lower with bactibilia (31 vs 68, P=0.07). Average slime production was 21 (+9.8) vs 45 (+6.7) for bacteria that did or did not cause bacteremia (P<0.0001), and no bacteria with slime production >75 demonstrated bacteremia. Bacteremia was more common if bG/PhLprod bacteria were present in the gallstone (18% vs 3%, with vs without bG/PhLprod, P=0.038). CONCLUSIONS: Bacteria-laden gallstones are biofilms whose characteristics influence bacterial colonization, detachment, and cholangiovenous reflux. Factors creating colonization surface(bG/PhL) facilitated bacteremia and severe infections; but abundant slime production, while facilitating colonization, inhibited detachment and cholangiovenous reflux. High slime-producing isolates were recovered from bile, but not blood. This underscores the liver’s filtering effect, which inhibited cholangiovenous reflux of high slime-producing bacteria. This data shows how the properties of the gallstone biofilm determine the severity of the associated clinical illness