Systemic antibody response to Clostridium difficile in colonized patients with and without symptoms and matched controls.

It has been proposed that patients who develop Clostridium difficile-associated disease (CDAD) do so because they are unable to mount an adequate immune response. Serum was collected from three groups of elderly in-patients: (i) cases (n=21) of CDAD, being toxin A/B-positive; (ii) carriers (n=21) asymptomatic for CDAD (no diarrhoea) but at least toxin or culture positive; and (iii) controls (n=26) asymptomatic for CDAD and negative for both C. difficile toxin and culture. The age and gender of each group were compared, and the colonizing strains were ribotyped and toxinotyped. Serum antibodies (IgG and IgM) were measured by ELISA using different antigen preparations: EDTA extract (containing cell-surface proteins and carbohydrates), guanidine hydrochloride extract (surface-layer proteins), aqueous phenol-extracted lipocarbohydrate (LC); crude toxin (dialysis culture supernatant) and purified toxin A. LPS from Escherichia coli was used as a control antigen. Antibodies were also tested for toxin neutralization on tissue monolayers and for binding to EDTA-extracted antigens by Western blotting. IgG antibody measurements to cytomegalovirus (CMV) were included as an indicator of potential immunosenescence. Results showed that the patient groups were well matched by age and gender, and the colonizing strains were similar in cases and carriers, being predominantly ribotype 001 and toxinotype 0. By ELISA, IgG levels to most of the antigens were highest in the cases and lowest in the controls, with the exception of antibodies to the LC, which were higher in the controls than the cases. Levels in the carriers tended to be of intermediate level or similar to the controls. For all antigens, the levels of IgM were not significantly different among cases, carriers and controls. Serum from all groups was able to neutralize the cytotoxic action of toxin on both Vero and Caco2 cells, and all to a similar extent. Western blots showed an overall higher level of IgG antibodies to the EDTA-extracted antigens in the cases. The results of the CMV ELISA showed that specific IgG was detected in more cases (78%) than carriers and controls (both 65%), but this difference in seropositivity was not significant. The conclusion is that, during symptomatic infection, patients respond to protein antigens of C. difficile in a manner typical of a secondary antibody response, with no evidence that an inability to respond predisposes to the appearance of symptoms.

Enhancement of the cytotoxic activity of Clostridium difficile toxin A by surface-associated antigens.

Cell-surface antigens of Clostridium difficile and LPS from Escherichia coli were investigated for modulating effects on the activity of C. difficile toxin A on Vero and Caco2 cells. The antigens of C. difficile tested comprised: (i) an EDTA extract, which contained several major and minor cell-surface proteins and the membrane-associated lipocarbohydrate (LC); (ii) a guanidine hydrochloride extract, which mainly contained the surface-layer proteins; (iii) an aqueous phenol-extracted, protein-free LC. On their own, none of the antigens had a detrimental effect on the cells, with the EDTA extract and LC having a marginally protective effect. When these antigens were added to suboptimal levels of toxin A, there was significant enhancement of its cytotoxicity by the EDTA and LC preparations on both cell types. LPS showed some enhancement of the effect of toxin on Vero cells at the lowest levels of toxin investigated. It was concluded that this effect seen in vitro may have a role to play in the colon during infection with C. difficile.

Molecular characterization and antimicrobial susceptibility patterns of Clostridium difficile strains isolated from hospitals in south-east Scotland.

Clostridium difficile isolates (n=149) collected in south-east Scotland between August and October 2005 were typed by four different methods and their susceptibility to seven different antibiotics was determined. The aims were to define the types of strain occurring in this region and to determine whether there were any clonal relationships among them with respect to genotype and antibiotic resistance pattern. Ribotyping revealed that 001 was the most common type (n=113, 75.8 %), followed by ribotype 106 (12 isolates, 8.1 %). The majority of the isolates (96.6 %, n=144) were of toxinotype 0, with two toxinotype V isolates and single isolates of toxinotypes I, IV and XIII. PCR and restriction analysis of the fliC gene from 147 isolates gave two restriction patterns: 145 of pattern VII and two of pattern I. Binary toxin genes were detected in only three isolates: two isolates of ribotype 126, toxinotype V, and one isolate of ribotype 023, toxinotype IV. S-types showed more variation, with 64.5 % (n=40) of the common S-type (4,939) and 21 % (n=13) of S-type 4,741, with six other S-types (one to three isolates each). All ribotype 001 isolates were of the same S-type (4,939), with three isolates of other ribotypes being this S-type. No resistance was found to metronidazole or vancomycin, with resistance to tetracycline only found in 4.3 % of the isolates. A high proportion of isolates were resistant to clindamycin (62.9 %), moxifloxacin, ceftriaxone (both 87.1 %) and erythromycin (94.8 %). Resistance to three antibiotics (erythromycin, clindamycin and ceftriaxone) was seen in 66 isolates, with erythromycin, ceftriaxone and moxifloxacin resistance seen in 96 isolates. Resistance to all four of these antibiotics was found in 62 isolates and resistance to five (the above plus tetracycline) in one isolate: a ribotype 001, toxinotype 0 strain. Whilst ribotype 001 was the most commonly encountered type, there was no evidence of clonal relationships when all other typing and antibiotic resistance patterns were taken into account.