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Enterococcal aggregation substance as target for opsonic antibodies in vitro and in murine sepsis


In ICU patients, the mortality of enterococcal sepsis is high, and treatment is often difficult due to multiple antibiotic resistance. Aggregation substance (AS) is a surface protein of E. faecalis, which mediates transfer of (resistance) plasmids and was also identified as a virulence factor. We investigated whether AS may serve as target for antibodies in phagocytosis assays and in a murine sepsis model.


Using the Qiagen pQE vector system, AS was expressed in E. coli and a histidine-tag served for purification by Ni-NTA chromatography. After purity was verified by western blot, AS was used to immunize rabbits. In phagocytosis assays, the immune rabbit sera (IRS) were compared with pre-immune sera for their opsonic activity, using human leukocytes, complement, and mutants of E. faecalis OG1. In order to show the specificity of the antibodies, the sera were absorbed with mutants of E. faecalis OG1 constitutively expressing (AS+), or not expressing AS (AS-). In the sepsis model, aliquots of IRS or normal rabbit sera (NRS) were used for IV passive immunization of Balb/c mice 24 hours before bacterial challenge, and 4 hours and 24 hours thereafter. IV bacterial challenge was performed via tail vein injection of AS+ E. faecalis. The mice were sacrificed 5 days thereafter for examination of bacterial levels in internal organs.


When incubated with IRS, the number of AS+ E. faecalis recovered at the end of the phagocytosis assay was reduced by more than 50% compared with incubation with pre-immune sera (P < 0.01), whereas there was no difference against AS- strains. IRS was still opsonic after absorption with AS- E. faecalis but the activity was lost after absorption with AS+ mutants. In the mouse sepsis model, weight loss was significantly less in the IRS group (mean ± SD, 97.9 ± 3.6% of baseline weight) versus the NRS group (95.3 ± 2.9%, P = 0.04). The highest organ load with enterococci were found in the kidneys and the log cfu/g were significantly lower in the IRS group (P = 0.03).


In our mouse sepsis model, antisera against AS significantly reduced weight loss and also the number of enterococci recovered from kidneys. The usefulness of AS as a target for antibodies is further confirmed by enhanced killing of AS+ enterococci by IRS. Our results are encouraging and point to the possibility of adjunctive immunotherapeutic approaches in enterococcal sepsis.

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Krueger, W., Escher, M., Woll, A. et al. Enterococcal aggregation substance as target for opsonic antibodies in vitro and in murine sepsis. Crit Care 7, P041 (2003).

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  • Normal Rabbit Seron
  • Bacterial Challenge
  • Aggregation Substance
  • Sepsis Model
  • Multiple Antibiotic Resistance