Introduction: Patients with decompensated cirrhosis are highly susceptible to bacterial infections, associated with compromised liver and immune function, and outcomes following infection in these patients are poor. Increasing antibiotic-resistant infections have been reported worldwide leading to emerging interest in host-directed therapeutic strategies. Ascites, an accumulation of fluid in the peritoneal cavity, is the commonest complication in patients with decompensated cirrhosis, and is the most common site of infection (Spontaneous Bacterial Peritonitis). Knowledge of ascitic fluid (AF) innate immune mechanisms and potential strategies to enhance immune function to prevent or treat infections is limited. This study investigated the bactericidal capacity of AF for E. coli, the commonest cause of SBP, and the mechanisms enabling a uropathogenic E. coli (UPEC) strain to resist AF killing.
Methods: AF killing assays were conducted with the non-pathogenic E. coli strain (MG1655) and the multidrug resistant UPEC ST131 strain EC958. Bacteria were incubated in cell-free AF for 90 minutes to assess survival. Deposition of complement and immunoglobulin on the E. coli strains was assessed by flow cytometry. Transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing (TraDIS) was used to identify genes required for EC958 survival in AF.
Results: E. coli MG1655 was susceptible to AF-mediated killing, but survived in heat-inactivated AF, suggesting an important contribution of complement. Consistent with this, robust deposition of complement C3 and the C5-9 membrane attack complex, but not C1q or IgG, on the surface of MG1655 was observed. In contrast, EC958 was resistant to AF-mediated killing and no complement deposition was observed. TraDIS analysis of an EC958 transposon mutant library identified 41 genes required for survival in AF. Similar to serum resistance mechanisms, many of these genes were related to capsule synthesis. Mutation of genes encoding the transcription factors RfaH, which controls capsule and lipopolysaccharide expression, and Fis, a nucleoid associated DNA-binding protein, rendered EC958 susceptible to AF killing, associated with restoration of complement C3 and C5-9 deposition.
Conclusion: AF has potent bactericidal capacity, mediated in part by the alternative complement pathway, however cell-surface components such as the capsule enable pathogenic E. coli to evade AF killing.