Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins

Abstract

A typing method for Clostridium difficile based on the incorporation of [35S]methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described. On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z). The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C. ditficile. The epidemiology of Clostridiium difficile diarrheal disease has not yet been fully clarified. Although C. difficile is known to be the primary cause of pseudomembranous colitis and antibiotic-associated colitis (AAC) and is associated with diarrhea after antibiotic therapy and chemotherapy (1, 8, 10, 11), this organism has also been isolated from the feces of healthy adults (0 to 11%) (1, 3, 12), from vaginal specimens of healthy mothers (18%) (18), and from asymptomatic infants and neonates (40 to 75%) (4, 7, 12, 15, 18). Although reports of clustering of cases and outbreaks of pseudomembranous colitis and AAC are suggestive of the nosocomial

Purification and Immunochemical Properties of a Wall Protein Antigen from Clostridium difficile ATCC 11011

“…These remarkable features were also found in the composition of antigen S of C. botulinum type A 190L (31). The molecular weight of the 32K antigen determined by sedimentation equilibrium (30,200) was fairly consistent with that estimated by SDS-PAGE (32,000), suggesting that the antigen molecule is a single polypeptide chain. The molecular weight of the 32K antigen is lower compared with the other RA proteins with molecular weights from about 41,000 to 200,000 (13,29).…”

“…Epidemiological studies have demonstrated that clustering of the cases and outbreaks of the colitis are due to the nosocomial spread of this organism (7,16,25). To date, various typing methods of C. difficile strains have been reported: Nakamura et al (20) classified them into four serovars by the crossed agglutination test using formol-treated cells; Delmee et al (6) defined six serogroups based on the slide agglutination; and Tabaqchali et al (30) grouped them into nine serovars by employing the electrophoresis of labeled cellular protein. However, immunochemical and physicochemical properties of the cellular components, which are responsible for these serological reactions, have not yet been thoroughly elucidated.…”

Numerical analysis of electrophoretic protein patterns of Providencia rettgeri strains from human faeces, urine and other specimens

“…Typing by PAGE overcomes such problems especially as the same approach can be used for other organisms (which for serotyping would each require a large range of sera). Other organisms to which PAGE typing has been applied include Acinetobacter (Alexander et al 1984), Bacteroides (Taylor et al 1987), Campylobacter (Costas et al 1987b), Clostridium (Tabaqchali et al 1986) and Streptobacillus .…”

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile

“…Nine standard strains of C. dlfficile, designated A-E and W-Z, were used [5,6] . The identity of the clostridal strains was confirmed by smell, colony morphology and gas-liquid chromatographic analysis of volatile fatty acids [7].…”

“…Three types of antigen were prepared for rabbit immunisations: i) C difficile whole cells from the 9 standard typed strains (A E and W-Z, identified by 35S-methionine labelled protein profiles [6]), were prepared from formalised cell antigens as described previously [5]. ii) E. coli lysates containing cloned C. difficile antigens were prepared from a confluent lawn of plaques on 10 plates (90 mm diameter) overlaid with 0.5 ml SM buffer (0.1 M NaC1, 10 mM MgSO4, 50 mM Tris (ph 7.5), 0.01% gelatine) for 1 h at 25 ° C. The plate lysate suspension was centrifuged (Hi-Spin 21 MSE Scientific Instruments, U.K.) at 7000 × g for 15 rain and the supernatant was used for rabbit immunisation.…”