Evaluation of methods for typing coagulase-negative staphylococci

Abstract

Summary.One hundred and forty-two coagulase-negative staphylococci (CNS) isolated from dialysate effluent or skin of patients receiving continuous ambulatory peritoneal dialysis (CAPD) were typed by extended antibiogram (16 antibiotics) and biotype (26 reactions). These isolates were then typed by supplementary methods to determine the most suitable typing method for an epidemiological study of antibiotic resistance. These included phage typing, reverse phage typing, plasmid typing, whole-cell protein typing by SDS-PAGE with analysis by densitometry, and immunoblotting. The percentage of isolates typed successfully by the supplementary methods were: phage typing 20 %, reverse phage typing 0 %, plasmid typing 66 %, SDS-PAGE 100 %, immunoblotting 100 YO. The discrimination of each method was : phage typing 20 YO, plasmid typing 37 %, SDS-PAGE 69 %, immunoblotting 57 YO.Reproducibility was 88 % for phage typing and 97 % for plasmid typing. The reproducibility of the whole-cell protein typing was 83% if the same extracts were used but only 43 YO when separate protein extracts were analysed on separate occasions. However, strain relatedness was highly reproducible. The determination of an antibiogram-biotype profile was not a sufficiently accurate typing method for an epidemiological study of antibiotic resistance. Whole-cell protein typing by SDS-PAGE or immunoblotting was technically demanding but was the most effective of the supplementary methods for detecting erroneous discrimination and false matching produced by antibiogram-biotype combinations.

Recommended minimal standards for description of new staphylococcal species

“…SDS-PAGE with Coomassie staining has produced distinct banding patterns for Staphylococcus species (Clink & Pennington, 1987). Dryden et al (1992) found that SDS-PAGE has a higher discriminatory power than globulin. Cultures grown in brain-heart infusion at 37 "C for 18 h are centrifuged and washed in 1/15 M phosphate buffer (pH 5.9).…”

Assessment of multiple coagulase-negative staphylococci isolated in blood cultures using pulsed-field gel electrophoresis

“…Intendente Jorge Palacios, 30003 Murcia, Spain. and plasmid profiles analyses (5). The only practical first-line methods to identify isolates of CNS in the routine laboratory, however, are the use of biotype profiles and antibiograms.…”

Serological response to coagulase-negative staphylococci in patients with peritonitis on continuous ambulatory peritoneal dialysis

“…A humoral response to CNS in serum and dialysate in CAPD patients has been detected by enzyme immunoassay, and CNS peritonitis has been shown to provoke a rise in antibody levels in some patients (13). Western immunoblotting, which we have adapted to type CNS (14), is a highly sensitive technique which may be employed to detect an antibody response to specific antigens. Immunoblotting has been employed to diagnose enterococcal endocarditis (15), and to investigate the antibody response of serum from healthy volunteers to Staphylococcus aureus (16).…”

“…Immunoblotting was carried out as described previously (14). Briefly, whole cell protein of the staphylococcal strains, extracted by sonication, was separated by SDS-PAGE in 10 % polyacrylamide gel in a discontinuous buffer system at 10*C. After transfer of the separated staphylococcal protein to the nitrocellulose membrane (0.45 ~tm membrane pore, Schleicher & Schuell, Germany) using 100 mA current for 15 h and blocking the free protein sites on the membrane with 5 % bovine serum albumin (BSA) in Tris buffered saline, the membranes were cut into pairs of lanes.…”