S. P. Borriello

Mucosal association by Clostridium difficile in the hamster gastrointestinal tract

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.

Vaccine potential of meningococcal FrpB: studies on surface exposure and functional attributes of common epitopes

Neisseria meningitidis expresses several novel outer membrane proteins (OMPs) in vivo and when grown under iron limitation in vitro. One of the most prominent is a 70 kDa iron-regulated protein (FrpB). FrpB was purified by elution from SDS-polyacrylamide gels and rabbit polyclonal antiserum (R-70) was raised against it. R-70 was bactericidal against homologous, but not heterologous, strains in the presence of human complement. The bactericidal activity was retained when R-70 was adsorbed with formaldehyde-fixed iron-replete cells (i.e. not expressing FrpB), but lost when absorbed with fixed iron-restricted cells (which express FrpB). A murine monoclonal anti-FrpB antibody (mAb M70) was raised against a common epitope which showed complete cross-reaction on Western blots of OMPs from other serogroups and serotypes of N. meningitidis and some commensal Neisseriae species. However, it failed to kill the organism. Immunogold electron microscopy on ultrathin sections, using the R-70 antiserum adsorbed with fixed iron-replete cells, showed labelling on 40% of the cells, whereas the R-70 adsorbed with fixed iron-restricted cells and mAb M70 failed to label. However, none of these sera labelled whole cells, suggesting lack of surface accessibility. It appears that the highly conserved cross-reactive epitopes of FrpB only become exposed in the process of generating the antigen, whereas the surface-exposed epitopes recognized in killing assays are immunologically variable among different strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity and cross-reactivity of the 70-Kda iron-regulated protein of Neisseria meningitidis in man and animals

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.

Characteristics of a gram-negative anaerobe isolated from men with non-gonococcal urethritis.

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.

Characterisation of anaerobic curved rods (Mobiluncus spp.) isolated from the urogenital tract

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.

Clostridium Perfringens Enterotoxin-Associated Diarrhoea

Enterotoxigenic strains of C. perfringens are now implicated in cases of diarrhoea other than food poisoning. There is good evidence that cross-infection may occur and that cross-infection control measures are helpful in limiting spread of the disease. The organism can be readily isolated from the immediate environment of affected patients as well as from their hands. In general the diarrhoea is self limiting, but metronidazole or vancomycin may be used to treat troublesome cases. Faecal blood and mucous and abdominal pain feature in about half of the cases.

Clostridium Difficile and Colonization Resistance

At birth the human intestine is sterile but very quickly becomes colonized with a wide variety of bacterial species, derived formerly from the mother and then from the environment. Eventually, the acquisition of new species ceases and the composition of the flora stabilises so that, in healthy individuals, host and microflora live in harmony. This relationship is important, for although the presence of an indigenous microflora is not essential for life, as is evident from the existence of gnotobiotic animals, it does exert an influence over the normal physiological functioning of the gastrointestinal tract. This includes control of fat absorption and cholic acid hydrolysis, metabolism of bilirubin, protein and urea into ammonia and the synthesis of vitamins from dietary substrate.

Abstracts of the First Meeting of the Society for Intestinal Microbial Ecology and Disease, Boston, Massachusetts, 1983

During enterohepatic circulation conjugated steroids are hydrolyzed by intestinal bacteria, further metabolized mainly by anaerobic bacteria, reabsorbed, reconjugated and delivered to the blood for renal excretion. 21-Dehydroxlylation of tetrahydrodeoxycorticosterone (THCOD) to pregnanolone is performed exclusively by Eubacterium lentum, a normal inhabitant of the intestinal flora. The concentration of E. lentum in feces can be determined from the highest dilution of fresh voided stool capable of 21-dehydroxylating THDOC. Such studies revealed that the concentration increases from 106/g wet feces during the first year of life to 108/g feces in the 7th and 8th decade. Colonization appears to be independent of diet since the organisms are present in babies whether breast-fed or on formula, in subjects on a “Western diet” and in Africans consuming a diet low in meat and fat.