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The soluble proteome of three Clostridium difficile strains of varying pathogenic potential, designated B-1, Tra 5/5 and 027 SM, were compared using differential in-gel electrophoresis in which the proteins of each strain were labelled with CyDyes. This enabled visual inspection of the 2D profiles of strains and identification of differentially expressed proteins using image analysis software. Unlabelled protein reference maps of the predominant proteins were then generated for each strain using 2D gel electrophoresis followed by protein sequencing of each spot using a Reflectron matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. Increased coverage of the proteome was achieved using 1D gel electrophoresis in a bottom-up approach using LC-MS/MS of 1 cm gel slices. A total of 888 different proteins were detected by comparative analysis of isolates grown in parallel for 64 h on blood agar plates. Of these, only 38 % were shared between all isolates. One hundred and ten proteins were identified as showing ≥2-fold difference in expression between strains. Differential expression was shown in a number of potential virulence and colonization factors. Toxin B was detected in the more virulent strains B-1 and 027 SM, but not in the lower virulent strain Tra 5/5, despite all strains possessing an intact pathogenicity locus. The S-layer protein (Cwp2) was identified in strains 027 SM and Tra 5/5 but not strain B-1, and differences in the post-translational modification of SlpA were noted for strain B-1. The variant S-layer profile of strain B-1 was confirmed by genomic comparison, which showed a 58 kb insertion in the S-layer operon of strain B-1. Differential post-translation modification events were also noted in flagellar proteins, thought to be due to differential glycosylation. This study highlights genomic and proteomic variation of different Clostridium difficile strains and suggests a number of factors may play a role in mediating the varying virulence of these different strains.

Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.

Summary Five well characterised strains of Clostridium difficile of differing virulence and two Escherichia coli strains, a verotoxigenic O157:H7 isolate and a urinary isolate, were examined for cell surface hydrophobicity and charge, and haemagglutinating ability. Phase partition in hexadecane or octan-1-ol was similar for C. difficile and E. coli, as was retention by hydrophobic interaction chromatography (HIC), indicating moderate hydrophobicity. The salt agglutination test showed E. coli to be hydrophobic and C. difficile to be hydrophilic. Relative hydrophobicity determined by HIC when charge effects were not nullified, i.e., to reflect more closely conditions in vivo, showed C. difficile to bind less well. Growth of C. difficile in caecal emulsions to simulate conditions in vivo did not alter the cell surface hydrophobicity. The phase partition method for charge determination indicated that E. coli and C. difficile had a net negative charge, although this was weaker for C. difficile than E. coli. However, although E. coli exhibited a net negative charge as determined by immuno-gold electronmicroscopy (IGEM), in keeping with the results of the phase partition method, C. difficile was shown to be predominantly positively charged by IGEM, and by movement in a charged field as determined by paper electrophoresis and a novel method based on light microscope observation. A cell-wall deficient mutant of C. difficile was weakly positively charged, showing that most of the charge resides in the cell wall.

Thirty isolates of Clostridium difficile expressing different degrees of toxigenicity and virulence in an animal model were assayed for the production of chondroitin-4-sulphatase, hyaluronidase, heparinase, collagenase and protease. All strains demonstrated some hydrolytic enzyme activity. There was no direct correlation between toxigenic status, or virulence, and hydrolytic enzyme production. However, all five strains known to be highly virulent in the hamster model had hyaluronidase, chondroitin-4-sulphatase and collagenase activity whereas only three of five toxigenic but poorly virulent strains had these activities, the collagenase activity being weak in all three cases. The only two proteolytic strains are also highly virulent. The potential tissue damaging properties of these hydrolytic enzymes may help to explain the differences in virulence of C. difficile strains seen in the Syrian hamster model of antibiotic-associated colitis, and may contribute to the spectrum of disease seen in man. It is also possible that chondroitin-4-sulphatase, hyaluronidase and collagenase activity may release essential nutrients, promoting establishment of C. difficile in the gut.

Summary A λZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative SDS-polyacrylamide gels. Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised. The cloned DNA insert was sequenced from positive clones and analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (Elp), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed as dihydrolipoyl acetyltransferase by immunological and biochemical assays. N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein. Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p. Conversely, rabbit antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity of this domain. Antibodies to Men-E2p were detected in patients and animals (rabbits and mice) infected with homologous or heterologous meningococci or other neisserial species. These results have important implications for the understanding of PDHC and the design of future outer membrane vesicle-based vaccines.

Iron-related tRNA alterations have been shown to occur in several pathogens but nothing has been reported about the effect of iron on meningococcal tRNA. The chromatographic elution profile of 3H-tryptophan-tRNA(trp) from a Neisseria meningitidis strain grown under different conditions was examined. The profile showed an early (P1) and a late (P2) eluting species of tRNA(trp), but the proportion of the two species varied under different growth conditions. The elution profile of trp-tRNA(trp) from bacteria grown in iron-sufficient Mueller Hinton broth yielded a minor P1 species and a major P2 species, whereas under iron-restricted growth induced by desferrioxamine, the pattern was one of a major P1 species and minor P2 species. Iron-restriction induced by human transferrin (HTF) resulted in almost equal amounts of the two tRNA(trp) species (P1 congruent to P2). Differences in the proportions of the tRNA species were also found between cells grown in liquid medium (P1P2) and on the same medium solidified with agar (P1P2). The growth phase of the bacteria did not have any effect on the tRNA(trp) elution profile. Changes in tRNA(trp) induced by HTF were readily and completely reversible when the cells were transferred to an iron-rich medium, but those induced by desferrioxamine remained irreversible for a long period (16 h) after such transfer.

Analysis of crude culture filtrate of Clostridium difficile by Mono Q-anion exchange fast protein liquid chromatography (FPLC) demonstrated that toxin A had distinct peaks of activity for cytotoxicity and haemagglutination, as also did highly purified toxin A obtained by thyroglobulin affinity chromatography (TG) followed by two sequential anion-exchange chromatographic steps with Q-Sepharose FF and Mono Q. From TG unbound fractions a highly cytotoxic but weakly haemagglutinating variant (toxin A') of toxin A was obtained by Q-Sepharose FF and Mono Q chromatography. Analysis of toxins A and A' from cultures of C. difficile in a chemically defined medium, and of toxin A dialysed against brain heart infusion broth, indicated that A' was not merely toxin A coupled to a component of the growth medium. Polyacrylamide gel electrophoresis under non-denaturing conditions showed that toxins A and A' had the same Mr. Immunoblotting with mouse monospecific A antitoxin showed that five bands larger than the major 240-Kda band were more strongly developed in toxin A than in A' in denaturing but non-reducing conditions, and in reducing conditions eight bands (38-175 Kda) were seen in toxin A but not A'. Immunoblotting with a monoclonal antibody (PCG-4) showed that, in both reducing and non-reducing conditions, two bands of 160 and 155 Kda were more prominent in toxins A and A' respectively, and four bands (195, 180, 175 and 125 Kda) were detected only in toxin A'.

On the basis of the above findings it is possible to propose a sequence of events following exposure to C. difficile. Exposure of neonates to C. difficile leads to transient colonisation which is almost invariably asymptomatic; the reasons why colonisation is asymptomatic are not known. Exposure of antibiotic-treated adults to C. difficile does not invariably lead to colonisation; however, in those instances where colonisation occurs, it may be transient and asymptomatic or transient and symptomatic. The transient nature of the colonisation could be because the infecting strain is poorly virulent, or because the degree of compromise of the intestinal flora is insufficient to permit establishment and full expression of virulence. It is likely that it is easier to fully compromise the intestinal flora of the elderly so that they more readily become fully susceptible to colonisation by C. difficile. In a fully susceptible host and with a highly virulent strain, the following sequence of events could occur. The organism may associate with the intestinal mucosa possible via fimbriae, and form a microcolony of capsulate cells protected by a glycocalyx. The toxins, or other factors, produced may facilitate the interaction with mucosa and toxin A will result in increased vascular and mucosal permeability resulting in intra-luminal accumulation of serum-albumin-rich fluid. Although C. difficile does not appear to be capable of using serum albumin nutritionally, it may utilise other serum proteins, and the serum proteins in general may compete with host proteases and help prevent degradation of the toxins produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary The immune response to different serogroups and serotypes of N. meningitidis has been examined in acute and convalescent sera from patients with meningococcal diseases. The focus of the study was the c. 70-Kda iron-regulated outer-membrane protein (FeRP-70). FeRP-70 was demonstrated on all strains of different serogroups and serotypes examined by sodium dodecylsulphate-polyacrylamide gel electrophoresis or Western blots of outer-membrane proteins (OMPs). Immunoblotting experiments demonstrated the presence of considerable amounts of anti-FeRP-70 IgG antibodies in the acute and convalescent sera of six patients; the antibodies reacted with homologous and heterologous strains. However, sera from two patients who died of severe meningococcal septicaemia had no antibodies against FeRP-70 or any other OMPs demonstrable by immunoblotting. Absorbed rabbit hyperimmune sera reacted with FeRP-70 of their homologous strains, but, unlike human sera, with only a few of the heterologous strains. We believe that FeRP-70 is strongly immunogenic in vivo, cross-reactive amongst different strains, and that man and animals differ considerably in their response to similar meningococcal antigens. The functional attribution of human antibody response against this protein requires further exploration.

SUMMARY. A small, fastidious gram-negative anaerobe was isolated from men with non-gonococcal urethritis (NGU). The isolates are described as NGU-associated anaerobes because they were extremely rare in men with urethritis other than NGU, and in asymptomatic men. They showed twitching motility, had many polar pili and appeared to be a homogenous group culturally, morphologically and biochemically. None of the strains fermented or utilised carbohydrates or organic acids as sole sources of carbon for energy and growth. However, growth of all strains was stimulated by formate and fumarate in liquid and solid media, especially in the former where growth seemed dependent on these growth factors. Unlike most anaerobes they produced cytochrome enzyme(s) that might be involved in oxidation-reduction reactions in the presence of oxygen as some of the strains were capable of growing in 5% oxygen. However, growth and energy generally resulted from anaerobic phosphorylation. Strains of this anaerobe seemed to require a low redox-potential (Eh) for survival during transportation but this was not essential for growth. Comparative studies with the other asaccharolytic anaerobes showed some similarity between the NGU-associated anaerobe, Bacteroides ureolyticus and Wolinella succinogenes. Like these, some NGU-associated strains pitted agar media and all produced urease. However, unlike these anaerobes, strains of the NGU-associated anaerobe produced enzymes for the hydrolysis of arginine, and the decarboxylation of lysine and ornithine. They also produced oxidase and some strains haemolysed sheep red cells. However, lactic acid was not an end-product of the metabolism of glucose by any of the strains. The NGU-associated anaerobes are strikingly different from anaerobic vibrios, B. praeacutus and B. asaccharolyticus.

Each of nine different toxigenic strains of Clostridium difficile was administered orally to groups of hamsters pre-treated with clindamycin and housed individually in sterile isolator boxes. Faecal pellets and caecal contents from well, diarrhoeic, moribund and freshly dead animals were analysed for C. difficile and toxins A (enterotoxin) and B (cytotoxin), and tissue obtained when animals were killed was examined histologically. Not all strains were equally virulent in this model. Four strains of C. difficile killed all animals within 48 h and are designated as highly virulent for hamsters. These strains were clinical isolates from three cases of disease in man and one case in a hamster. Five strains caused death of some animals but only after 5 and upt to 13 days and are designated as less virulent for hamsters. These strains were isolated from asymptomatic infants (2) and household pets (2), and from the environment (1). The surviving test hamsters were killed after 14 days and, in most cases, were colonised by C. difficile, though levels of toxins A and B in caecal contents were low. None of the cultures used for challenge was capsulate or hydrophobic. There was no correlation between virulence and production of toxins A and B in vitro in tryptic-nitrate broth. With two strains examined, there was a correlation between virulence and toxin A (but not toxin B) production in caecal emulsions derived from clindamycin pre-treated hamsters. Caecal contents from the majority of moribund and freshly dead animals had quantities of toxin A sufficient to cause disease or death if given orogastrically. Toxin B was not produced in a fixed ratio with toxin A. The data support the view that high virulence of C. difficile is determined by efficient disease-inducing colonisation of the gut and the ability to generate, rapidly, high levels of toxin A in vivo.

Thirty-two strains of anaerobic curved rods isolated from vaginal secretions and one isolated from seminal fluid were examined. Growth of all strains on solid media was superior to growth in liquid media, and at 37 degrees C they grew both anaerobically and in O2 5% in N2; they also grew anaerobically at 33 degrees C but not at 42 degrees C. No growth factors were identified, but strains grew more profusely at pH values above 5 X 0. The strains were screened in 80 biochemical tests, and for their susceptibility to 30 different antimicrobial agents. Most of the tests did not differentiate between the strains, but they were divided into four groups on the basis of cell morphology, metronidazole susceptibility, beta-galactosidase activity and arginine and hippurate hydrolysis. Group 1 consisted of 19 strains conforming to the species M. curtisi; group 2 consisted of five strains conforming to the species M. mulieris; group 3 consisted of five strains that resembled M. curtisi morphologically, and group 4 consisted of four strains that resembled M. mulieris morphologically, but the strains in the latter two groups reacted differently in at least one of the three major differential biochemical tests. Of three strains of M. curtisi and three of M. mulieris chosen at random, one of M. mulieris had a SDS-PAGE and fast-protein liquid chromatography protein profile indistinguishable from that of M. curtisi. We conclude that further efforts are required to clarify the taxonomic status of the genus Mobiluncus.

Summary. Prior colonisation of clindamycin-treated hamsters with non-toxigenic strains of C. difficile protected them from subsequent colonisation with a toxigenic pathogenic strain. In total, 13 of 18 ‘protected’ hamsters survived for up to 27 days whereas all 27 animals challenged with the toxigenic strain alone died within 48 h. Protection was not evident if a heat-killed suspension was used or if the colonising non-toxigenic strain was first removed with vancomycin. No antitoxic activity could be detected in the faeces of animals colonised with the non-toxigenic strains. Other species of clostridia did not protect against the lethal effects of subsequent exposure to the toxigenic strain. Conversely, non-toxigenic strains would not protect the animals from the lethal effects of a different clostridial pathogen, C. spiroforme. In most cases, even in the protected animals, the toxigenic strain eventually became dominant and caused disease, with translocation across the gut wall occurring early in the disease process. It was also shown that a non-toxigenic strain of C. difficile can adhere to gut mucosa. It is proposed that the protection afforded by the non-toxigenic strains may be due to competition for ecological niches.

To investigate the importance of the normal gut flora in preventing the establishment of Clostridium difficile in vivo we have developed an in-vitro test system based on growth in faecal emulsions. Growth of C. difficile and cytotoxin production are inhibited in faecal emulsions from healthy adults, but not in sterilised emulsions; the importance of viable bacteria in the inhibitory system is evident. Generally, faecal emulsions derived from infants, children and geriatric patients were less inhibitory than those from healthy adults. Those from bottle-fed infants were significantly less inhibitory than those from breast-fed infants. Decreased levels of cytotoxin in the latter group were attributed to the acidic pH of the stools. With the different patient groups studied, faecal samples not inhibitory to C. difficile in vitro were obtained from 21% of patients with antibiotic-associated diarrhoea, 33% of those taking antibiotics but who did not have diarrhoea, 18.7% of those with diarrhoea unassociated with antibiotics, and 79% of those with C. difficile-mediated diarrhoea. In some cases inhibition was due to low faecal pH, as in some infants, and in others to other filterable substances. The degree of inhibition could not be linked to specific volatile fatty acids or enzymes.

Highly purified toxin A of Clostridium difficile was obtained by bovine thyroglobulin affinity chromatography followed by two sequential anion-exchange chromatography steps on Q Sepharose FF and Mono Q. After Q Sepharose FF chromatography of a thyroglobulin affinity-purified toxin A preparation, two major peaks of cytotoxicity representing toxins A and B were detected. The homogeneity of the final toxin A preparation obtained from Mono Q anion-exchange fast protein liquid chromatography was ascertained by gel electrophoresis developed by silver stain. The mol. wt of toxin A in non-denaturing conditions was estimated to be 520-540 Kda by native polyacrylamide gel electrophoresis (PAGE) developed by silver stain. In contrast, with sodium dodecyl sulphate (SDS)-PAGE under reducing or non-reducing conditions, a major band of 240 Kda and 10 minor and 27 faint bands (non-reduced conditions), or four minor and 31 faint bands (reduced conditions) were detected after silver staining. In two-dimensional PAGE, the seven minor bands of greater than 240 Kda obtained by non-reducing SDS-PAGE migrated to the 240-Kda position after reduction with beta-mercaptoethanol.

For many organisms, mucosal association is an important virulence determinant. Although studied in detail for other intestinal pathogens, this aspect of pathogenicity has not been studied for Clostridium difficile. We compared the ability of an avirulent non-toxigenic strain (M-1), a highly virulent toxigenic strain (B-1), and a poorly virulent toxigenic strain (BAT) of C. difficile to adhere to different regions of the gastrointestinal tract of hamsters pre-treated with clindamycin. Strain B-1 associated with the gut mucosa significantly better than strain M-1 (p less than 0.001) for all sites other than the caecum, and achieved significantly higher levels in the caecal contents (p less than 0.001). The same was true when strain B-1 was compared with strain BAT except that there was no significant difference for the large bowel mucosa. To assess the possible role of toxin in promoting mucosal association, e.g., by compromising host defences or exposing masked adherence sites, strain M-1 was given to animals after intra-caecal administration of crude toxin preparations from strain-B1, which were heat-inactivated in control experiments. The addition of this toxin increased significantly the mucosal association of M-1 for the small bowel only, whereas the inactivated toxin had no significant effect. These results imply that there may be intrinsic differences between strains in their ability to colonise and associate with the gut mucosa, which may partly depend on their ability to produce toxin. These differences do not correlate with cell-surface hydrophobicity or the presence of plasmids, flagella or fimbriae.

Summary Enterotoxigenic strains of Clostridium perfringens have recently been implicated in some cases of antibiotic-associated diarrhoea. We present here the results of an epidemiological study of this disease. Five cases of diarrhoea caused by C. perfringens serotype 41 occurred during a 9-week period, and then during a 6-week period there were three cases due to serotype 27 and two due to serotype 24; in all but one case two geriatric wards were involved. In total there were 16 cases in 22 months. All cases were identified by the detection of C. perfringens enterotoxin in the faeces. The mean number of C. perfringens in these cases was 108.8 cfu/g of faeces. Of 37 patients who had negative test results for C. perfringens enterotoxin, 18 had positive cultures for C. perfringens, with mean faecal counts of 105.3 cfu/g, and nine of these patients had diarrhoea. Thirteen different serotypes were isolated from these 18 patients, including type 41 from seven patients and type 27 from one. Hand carriage of the offending serotype was demonstrated in three of four infected patients, none of four controls and two of 14 ward staff. C. perfringens of serotypes causing disease was isolated from 59% of environmental areas where there was active disease, 27% of areas where there had been disease which had since resolved and 9% of areas where there was no history of disease. The findings imply that cross infection may occur.

We compared the performance of a locally produced β-mercaptoethanol-modified promastigote antigen (β-ME-Ag) of an indigenous Leishmania infantum strain against that of a trypsinized Leishmania donovani reference (REF-Ag) in the direct agglutination test (DAT) for detection of canine visceral leishmaniasis (CVL). One hundred and fifty-one serum samples collected from dogs belonging to four groups with different conditions were included. At a DAT titre of 1 : 320, statistically determined as optimal cut-off value for β-ME-Ag, and 1 : 160 for REF-Ag, a sensitivity and a specificity of 100 % were estimated for β-ME-Ag in comparison with 96.6 % and 100 %, respectively, for REF-Ag. Overall, levels of agglutination titres recorded for the two antigens were highly concordant (Cohen’s κ = 0.879) in both the CVL and non-CVL groups. Based on current results, and ease experienced in processing the antigen and reading the test outcome, we recommend incorporation of β-ME-Ag in DAT for confirmation or exclusion of suspected CVL in dogs.