A Shared noncapsular antigen is responsible for false-positive reactions by Staphylococcus epidermidis in commercial agglutination tests for Staphylococcus aureus

Abstract

Many of the commercial slide agglutination tests for Staphylococcus aureus incorporate antibodies against cell surface antigens associated with methicillin resistance, including capsular polysaccharides and an uncharacterized antigen, serotype 18. These tests are more sensitive than the first-generation agglutination procedures that detected only bound coagulase and protein A, but they suffer from false-positive reactions with some coagulase-negative staphylococci. The aim of this study was to elucidate the mechanism for false-positive agglutination by S. epidermidis in these tests. A group of methicillin-resistant S. aureus (MRSA) isolates, including a serotype 18 strain, that were not detectable in the first-generation tests were found to be of capsular polysaccharide type 8. All of these isolates were deficient in bound coagulase and/or protein A, and they possessed a heat-stable, proteinaceous antigen that was absent from a prototype capsule type 8 strain. Enzyme-linked immunosorbent assay and agarose gel immunodiffusion experiments demonstrated that this proteinaceous antigen was also present on both methicillin-sensitive and methicillin-resistant S. epidermidis clinical isolates. S. epidermidis strains that gave false-positive agglutination test results had a considerably higher level of this antigen than strains that gave the correct negative result. These findings reveal the importance of the careful selection of MRSA strains for raising anti-capsular type 8 antibodies for use in agglutination tests. Strains devoid of the antigen shared with S. epidermidis should be used to eliminate potential cross-reactions with this coagulase-negative coccus.

FIG. 1

Assay of protein A in live cell suspensions (A) and extracts (B) of S. aureus strains Cowan (■), Wood (●), type 5 (⧫), type 8 (▴), MRSA1 (□), MRSA2 (○), MRSA3 (◊), serotype 18 (▵), and S. epidermidis ATCC 12228 ().

FIG. 2

Serotyping of S. aureus and S. epidermidis isolates by ELISA. Rabbit sera were as follows: anti-nonencapsulated S. aureus Wood serum (■), diluted 1:1,000; anti-type 5 (□) and anti-type 8 (▧) sera, diluted 1:2,000; and anti-serotype 18 serum (▨), diluted 1:4,000. All determinations were done in duplicate.

FIG. 3

Ability of bacterial extracts to inhibit the binding in an ELISA of anti-type 8 serum to type 8 cells (A), of anti-serotype 18 serum to serotype 18 cells (B), and of anti-serotype 18 serum to a problematic S. epidermidis strain, 2213 (C). Extracts of S. aureus strains were nonencapsulated (⧫), type 8 (▴), serotype 18 (●), and MRSA3 (■). Extracts of S. epidermidis strains were 2214 (○) and 2213 (□). Serum dilutions are given in the legend to Fig. 2. All determinations were done in triplicate.

FIG. 4

Agarose gel immunodiffusion analysis of bacterial extracts (1 to 12) with the following absorbed sera: anti-type 8 serum, unconcentrated (A); anti-serotype 18 serum, concentrated threefold (B to D); and anti-S. epidermidis 2213 serum, concentrated threefold (E). S. aureus extracts were as follows: 1, type 8; 2, serotype 18; 3, MRSA1; 4, MRSA2; and 5, MRSA3. S. epidermidis extracts were as follows: 6, 2034; 7, 2038; 8, 2213; 9, 2216; 10, 2222; 11, 2227; and 12, 2214. All extracts were diluted 1:2 in PBS.