Serological and genetic diversity of capsular polysaccharides in Enterococcus faecalis

Abstract

Enterococci possess capsular carbohydrate antigens that are targets of opsonic antibodies. These antigens may be used to develop alternative options for the treatment and prevention of enterococcal infections. The present study was done to analyze the diversity of capsular polysaccharides in Enterococcus faecalis. Four type-specific sera were used in an enzyme-linked immunosorbent assay format to detect polysaccharide antigen extracted from bacterial cell walls. A total of 55% of a collection of 29 E. faecalis strains could be grouped into one of four serogroups. Additional analysis of the strains by opsonophagocytic assays revealed agreement between the results of the two methods for 72% of the isolates. An additional four strains could be assigned to a serogroup on the basis of opsonic killing by sera with antibodies against the four prototypes strains, provisionally named CPS-A to CPS-D. The results of the two methods disagreed for one strain (4%). When the results of both methods were combined, 66% of the strains could be classified. One strain had to be assigned to two serogroups. The assignments to the four serogroups were confirmed by analysis of the genetic organization of the biosynthetic capsular polysaccharide (cps) locus. All strains grouped into serotypes CPS-A and CPS-B possessed only the cpsA and cpsB genes, while all strains grouped into serogroups CPS-C and CPS-D possessed an additional eight or nine genes. Our results suggest the existence of a limited number of E. faecalis capsule serotypes, and we provisionally propose four serotypes, named CPS-A to CPS-D, and the respective prototype strains for these families.

FIG. 1.

Immunoelectron microscopy of the four prototype E. faecalis (EFS) strains labeled with the prototype serum absorbed with either the homologous strain or the three heterologous strains.

FIG. 2.

Absorption CPS-ELISA of E. faecalis (EFS) type 2 and type 5 prototype strains with the two corresponding prototype sera. The two types of sera were absorbed either with the respective homologous strain or with each of the three heterologous strains. The percent absorption values reflect the ratio of the amount of absorbed sera to the amount of unabsorbed sera. Both types of sera could be absorbed with the homologous strain (84 and 91%, respectively). Absorption of both types of sera with heterologous strains E. faecalis type 2 and E. faecalis type 5, respectively, led to a higher degree of absorption (57 and 83%, respectively) than absorption with the heterologous strains E. faecalis 12030 and E. faecalis 12107 (range, 11 to 22%).

FIG. 3.

Phagocytic inhibition assay of the four prototype groups of sera against the four prototype E. faecalis (EFS) strains. Each prototype serum was tested against its homologous prototype E. faecalis strain by absorption with the respective homologous strain and with the three heterologous strains. The percent inhibition values reflect the ratio of the amount of absorbed sera to the amount of unabsorbed sera. The killing activities of the four types of sera were most effectively inhibited by absorption with the homologous strain (100% for E. faecalis 12030, 84% for E. faecalis 12107, 66% for E. faecalis type 5). Type 2-specific sera could not be absorbed to the same degree (absorption, 34%). Inhibition of killing activity by heterologous strains was greatly reduced for all four prototype sera, with cross-reactivity between strains E. faecalis 12030 and E. faecalis 12107.

FIG. 4.

Southern blot analysis of capsular polysaccharide determinants from various E. faecalis strains. DNA was restricted with ClaI and probed with the radiolabeled fragment of the cpsA to cpsK genes from E. faecalis strain V583. Lanes 1 to 5, strains with RFLP pattern I; lanes 6 to 10, strains with RFLP pattern II; lanes 11 to 15, strains with RFLP pattern III. Lane 1, E. faecalis 12107; lane 2, E. faecalis 12030; lane 3, E. faecalis type 4; lane 4, E. faecalis type 1; lane 5, E. faecalis type 7; lane 6, E. faecalis type 6; lane 7, E. faecalis type 5; lane 8, E. faecalis type 18; lane 9, E. faecalis type 14; lane 10, E. faecalis type 12; lane 11, E. faecalis type 8, lane 12, E. faecalis type 2; lane 13, E. faecalis MMH 594; lane 14, E. faecalis V583; lane 15, E. faecalis FA2-2. The sizes (in base pairs) of the E. faecalis V583 sequence fragments hybridizing to the probe are shown at the left. The ClaI restriction map for the E. faecalis V583 capsular polysaccharide determinant is shown below. The molecular sizes (in base pairs) of the regions spanned by the ClaI sites in E. faecalis V583 are listed below the map.

FIG. 5.

Comparison of E. faecalis strains FA2-2 and HG101 in the opsonophagocytic assay with NRS and the type 2-specific sera. Negative killing reflects growth during the 90-min incubation period of the assay.