Search

We evaluated clinical and diagnostic indicators of severe C. difficile infection (CDI) and their association with poor clinical outcome. A total of 210 patients positive according to PCR (toxin B: tcdB) were included, with patients having a median age of 62 years and a Charlson co-morbidity index (CI) score of 5. Ninety-one percent (n = 191) were positive by toxigenic culture and 61 % (n = 129) had stool toxin. Toxin-positive patients had significantly higher fecal lactoferrin (mean 316 μg/g versus 106 μg/g stool; p < 0.0001). Forty percent of patients (n = 85) were infected with ribotype 027 and significantly more of these patients had measurable stool toxin (79 % vs. 50 %; p < 0.0001). The mean fecal lactoferrin was significantly higher for toxin-positive 027 CDI compared with the 027 toxin-negative group (317 vs 60 μg/g; p = 0.0014). Ribotype 027 CDI with stool toxin showed a higher all-cause, 100-day mortality compared with non-027 with stool toxin (36 % vs 18 %; p = 0.017). Logistic regression univariate analysis for odds ratio (OR) and p values revealed that age (OR = 1.1), intensive care unit treatment (OR = 2.7), CI (OR = 1.2), 027 CDI (OR = 2.1), white blood cell count (OR = 1.0), albumin level (OR = 0.1), and stool toxin-positive 027 CDI (OR = 2.5) were significantly associated with 100-day mortality (p < 0.05). In conclusion, CDI PCR-positive patients with 027 infection and stool toxin have increased lactoferrin and are at an increased risk of death.

We evaluated blood and fecal biomarkers as indicators of severity in symptomatic patients with confirmed Clostridium difficile infection (CDI). Recruitment included patients with CDI based on clinical symptoms and supporting laboratory findings. Disease severity was defined by physician’s assessment and blood and fecal biomarkers were measured. Toxigenic culture done using spore enrichment and toxin B detected by tissue culture were done as confirmatory tests. Polymerase chain reaction (PCR) ribotyping was performed on each isolate. There were 98 patients recruited, with 85 (87 %) confirmed cases of toxigenic CDI (21 severe, 57 moderate, and seven mild), of which 68 (80 %) were also stool toxin-positive. Elevated lactoferrin (p = 0.01), increased white blood cell (WBC) count (p = 0.08), and low serum albumin (p = 0.03) were all associated with the more severe cases of CDI. Ribotype 027 infection accounted for 71 % of severe cases (p

We evaluated Clostridium difficile prevalence rates in 2,807 clinically indicated stool specimens stratified by inpatient (IP), nursing home patient (NH), outpatient (OP), age, gender, and specimen consistency using bacterial culture, toxin detection, and polymerase chain reaction (PCR) ribotyping. Rates were determined based on the detection of toxigenic C. difficile isolates. We identified significant differences in the rates between patient populations and with age. Specimens from NH had a higher rate (46%) for toxigenic C. difficile than specimens from IP (18%) and OP (17%). There were no gender-related differences in the rates. Liquid specimens had a lower rate (15%) than partially formed and soft specimens (25%) and formed specimens (18%) for the isolation of toxigenic C. difficile. The nontoxigenic rate was lowest for NH (4%) and highest for patients

Commercial rabbits showing clinical signs of enteritis-complex were examined for the presence of Clostridium spiroforme and its iota-like toxin. The bacterium was detected by Gram stain in 52.4% of 149 cecal samples and iota-like toxin in 7.4%. From 29 strains of C. spiroforme tested, 26 were toxigenic, originating from 24 of 29 rabbitries. In 13.4% of the samples, C. spiroforme was present as the only known disease agent. Gross and microscopic lesions were similar to those described in the literature. In the other samples, C. spiroforme was associated with attaching effacing Escherichia coli (29.5%), Bacillus piliformis (10.3%), rotaviruses (25.6%), coronavirus (2.6%), Eimeria spp. (44.9%) and cryptosporidia (6.4%). In 33.3% of C. spiroforme-containing samples, more than one of these agents was present. There was no significant difference between the presence of these organisms in C. spiroforme-positive and negative samples. On the basis of these results as well as that of previous data, we suggest that C. spiroforme-mediated diarrhea is favoured by maldigestion, initiated by infectious agents and/or nutritional factors.

The present study examined the IgG antibody activity in sera from a case:control population against whole cell proteins of Porphyromonas gingivalis and four other presumed periodontal pathogens: Bacteroides intermedius ; Capnocytophaga ochracea ; Wolinella recta ; Fusobacterium nucleatum . Whole cell enzyme-linked immunosorbent assays (ELISA) were performed to provide quantitative data on levels of IgG antibody and immunoblotting of whole cell lysates was carried out on 10 case:control pairs to identify the major individual antigens of each organism. For all organisms the mean level of IgG antibody activity against whole cells was higher in the cases compared to controls. For P. gingivalis the difference was highly significant ( p

Serum from adults with and without evidence of past periodontitis was assayed for the presence of antibodies to outer membrane components of Porphyromonas gingivalis . A protein of 47 000 Da in Sarcosyl-insoluble outer membrane preparations was shown to be a discriminatory antigen insofar as serum from 29 of 31 (94 per cent) patients with a history ofchronic adult periodontitis contained immunoglobulin G antibodies against this protein compared to four of 24 (17 per cent) serum samples from individuals without disease. The binding of antibodies to this antigen was shown to be F(ab’) 2 mediated. Type and reference strains of other oral and intestinal anaerobic bacteria were studied to see if the immunoreactive protein of 47 000 Da was exclusive to this species. None of the other microorganisms tested possessed this antigen, indicating that it is specific to P. gingivalis . Keywords: Porphyromonas gingivalis ; Bacteroides gingivalis ; Periodontal disease.

Staphylococcus aureus is a facultative, Gram-positive coccus well known for its disease-causing capabilities. In particular, methicillin and vancomycin resistant strains of S. aureus (MRSA and VRSA, respectively) isolated globally represent daunting medical challenges for the 21(st) Century. This bacterium causes numerous illnesses in humans such as food poisoning, skin infections, osteomyelitis, endocarditis, pneumonia, enterocolitis, toxic shock, and autoimmune disorders. A few of the many virulence factors attributed to S. aureus include antibiotic resistance, capsule, coagulase, lipase, hyaluronidase, protein A, fibronectin-binding protein, and multiple toxins with diverse activities. One family of protein toxins is the staphylococcal enterotoxins (SEs) and related toxic shock syndrome toxin-1 (TSST-1) that act as superantigens. There are more than twenty different SEs described to date with varying amino acid sequences, common conformations, and similar biological effects. By definition, very low (picomolar) concentrations of these superantigenic toxins activate specific T-cell subsets after binding to major histocompatibility complex class II. Activated T-cells vigorously proliferate and release proinflammatory cytokines plus chemokines that can elicit fever, hypotension, and other ailments which include a potentially lethal shock. In vitro and in vivo models are available for studying the SEs and TSST-1, thus providing important tools for understanding modes of action and subsequently countering these toxins via experimental vaccines or therapeutics. This review succinctly presents the pathogenic ways of S. aureus, with a toxic twist. There will be a particular focus upon the biological and biochemical properties of, plus current neutralization strategies targeting, staphylcoccocal superantigens like the SEs and TSST-1.

We have reviewed the recent literature on the humoral immune responses to a variety of subgingival plaque bacterial species in patients with destructive periodontal diseases. We do not feel that the information presently available on the specific antibody responses to proposed pathogens such as Bacteroides gingivalis and Actinobacillus actinomycetemcomitans allows antibody responses to be diagnostic. All control subjects without periodontal destruction have antibodies to candidate pathogens but the generally higher levels in patients are not sufficiently elevated to be diagnostic. Nor can they be used to predict the initiation of disease or the onset of new episodes of destruction where disease had previously occurred. Successful treatment of patients may lead to lower levels of antibodies to some organisms, including possible pathogens, and thus support a given species in the aetiopathogenesis of disease. It appears that unsuccessful treatment may be accompanied by continuing high antibody levels to some organisms and further studies may enable this observation to be used to monitor therapy. There is some evidence from serological studies that each destructive episode may be induced by a different bacterial species or consortium. The start of studies using single antigens and the techniques of molecular biology will provide not only antibody-based diagnostic methods but also allow us to determine which bacterial antigens are virulence factors and thus the role of the antibody responses, whether protective or damaging, in the periodontal diseases.

Clostridium difficile and Clostridium spiroforme have only in recent years been recognized as intestinal pathogens. They both produce toxins that are also produced by other clostridia. C. difficile toxins A and B are produced by C. sordellii and a few strains of C. perfringens whereas C. spiroforme produces the same toxins as C. perfringens Type E (iota toxin). Iota toxin activity may be the product of two proteins. Toxigenic strains of C. spiroforme and Type E produce two antigens which possess much more biological activity when administered together than when given alone. C. difficile was thought for some time to produce only a single toxin, but then the enterotoxic activity was shown to be due to a separate toxin (toxin A). This toxin increases the oral toxicity of toxin B (the main cytotoxin) and may increase the permeability of the colon. Toxin A binds to a specific receptor in hamster brush border membranes and in the membranes of rabbit erythrocytes. This receptor appears to be a glycoprotein. The receptor can be extracted from the membrane with Triton and binds to immobilized toxin A. The receptor can be extracted and used to coat plastic plates as a first phase in an ELISA assay. Another assay has been developed in which the toxin A binds to the red cells and then the erythrocytes are agglutinated with antitoxin. An even more sensitive assay consists of using rabbit erythrocyte ghosts to bind the toxin and then precipitating the ghosts with antibody to toxin A attached to latex beads. Monoclonal antibodies to toxin A also have been developed and are used in these and other assays.

Washed cells and Sarkosyl-insoluble outer membrane preparations of the black-pigmented bacteroides Porphyromonas gingivalis W50 bound hemin. The amount of hemin removed from a buffered solution by both cells and outer membranes was significantly larger if bacteria had been grown in broths supplemented with 5 mg of hemin per liter rather than none. Conversely, cells grown without supplemental hemin bound relatively little. However, all preparations bound some hemin. In addition, hemin regulated the production of significantly higher levels of trypsinlike protease by P. gingivalis W50. The nonpigmented variant, W50 BE1, showed no such responses to the levels of hemin in the growth medium.

The histological evaluation of surgical biopsies from affected tissues is a standard way of assessing pathological change and determining treatment in many diseases. In most forms of periodontal disease, however, this approach finds limited application. Here, we review what uses the histopathological approach has in the study and evaluation of the periodontal diseases. Current understanding of the changes in epithelial anatomy during pocket formation, the cellular composition and dynamics of the inflammatory infiltrate and the mechanisms of bone resorption and repair are reviewed from the perspective of the information available from microscopical investigation, including the uses and potential application of modern immunocytochemical methods to these questions. The usefulness of histological study of biopsy material is reassessed in the light of advances made in immunohistochemical techniques and their application to gingival inflammatory infiltrates and epithelia. Such techniques offer immediately valuable research opportunities with potential for diagnostic applications, noteably the recognition of phases of destructive activity and their differentiation from periods of effective host defence.

Periodontal microbiology is reviewed with regard to the potential of certain characteristics to serve as markers of high risk groups or individuals for periodontal diseases. The generally accepted associations between particular organisms and the various periodontal diseases are discussed. The usefulness of various clinical study designs is reviewed. The ecology of the subgingival plaque microflora is discussed and a number of suggestions for future research are made. We have concluded that there is no monospecific aetiology to any of the various periodontal conditions. Nevertheless, we give particular attention to the role of the black-pigmented bacteroides based upon our belief that they, and Bacteroides gingivalis in particular, are fundamental to our understanding of the biology of periodontal diseases in humans and other animals. Consequently, the contribution of its various virulence factors and their potential as markers of disease susceptibility and activity is addressed.

Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. “Candidatus Arthromitus” is proposed as the provisional generic name for this group of organisms.

The small-subunit rRNA (16S rRNA) sequence of Tyzzer's bacillus (also known as “Bacillus piliformis”) was elucidated by using the polymerase chain reaction followed by reverse transcriptase sequencing. By using maximum-likelihood analysis, a phylogenetic tree was constructed from this and other 16S rRNA sequences available from the first release of the Ribosomal Database Project (G. J. Olsen, R. Overbeek, N. Larsen, T. L. Marsh, M. J. McCaughey, M. A. Maciukenas, W.-M. Kuan, T. J. Macke, Y. Xing, and C. R. Woese, Nucleic Acids Res. 20:2199-2200, 1992). Tyzzer's bacillus grouped with a specific set of anaerobic bacteria, most of which are Clostridium spp. The closest identified relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii. Clostridium amino-valericum and “Acetitomaculum ruminis” are also solidly allied with this ensemble. We propose that Tyzzer's bacillus be reclassified as Clostridium piliforme on the basis of its 16S rRNA sequence.

The composition and functions of the intestinal floras from humans and other animals is compared and contrasted. Intrinsic and extrinsic factors, such as anatomy, age, and diet, help define the nature of the flora typical to each species of animal. Stable differences, such as the ability to reduce cholesterol or not, between the flora of individuals of the same species are described. The difficulties of characterizing a flora by cultural taxonomy are discussed and alternatives are suggested. These alternatives, collectively known as microflora associated characteristics (MACs), are measures of the biochemical activity of the bacterial flora. How MACs can be used to measure the effects of subtherapeutic levels of antibiotics on a flora is described.

Using pump sources of 1.064μm, greater than 500mW of CW power at 1.115μm has been measured from an Ytterbium Fibre Laser. Fibre Bragg gratings have also been used to obtain lasing at 1.128μm.

The identity of the major surface antigens of Porphyromonas gingivalis was investigated. Outer membranes of P. gingivalis strains W83, W50, 381 and NCTC 11843 were prepared following inactivation of the trypsin-like enzyme activity. Three proteins, molecular weight 115, 55 and 40 kDa, were major components of the outer membranes of strains W83 and W50 and were also present in strains 381 and NCTC 11834. Two proteins, 55 and 47 kDa, were released from the cells during the sonication step of the outer membrane preparation procedure. Immunoblots using preparations of P. gingivalis W83 and serum from a case-control study of adult periodontitis demonstrated higher mean antibody reactivity in the case population to all the major proteins except for the 115 kDa outer membrane protein, which was recognized equally well by both populations. We conclude that the 55, 47 and 40 kDa proteins are important surface antigens of P. gingivalis. Characterization of the structure and function of these components should lead to an improved understanding of the host-parasite interactions in adult periodontitis.

Carman, R.J. and Wilkins, T.D., 1991. In vitro susceptibility of rabbit strains of Clostridium spiroforme to antimicrobial agents. Vet. Microbiol., 28:391-397. Using an agar dilution method we measured the minimum inhibitory concentration (MIC) of 12 antimicrobial agents against 11 strains ofiota-toxigenic strains of Clostridium spiroforme. Each strain was isolated from a separate outbreak of toxic diarrhoea of rabbits. Vancomycin and bacitracin, both agents used to treat intestinal clostridioses of humans and other animals, had a relatively high MIC (8/~g/ml or more ). Metronidazole was uniformly active against C. spiroforme. With M1C of 8/~g/ml or more, both lincomycin ( 11 strains) and erythromycin (9 strains) were relatively inactive against C. spiroforme; conversely, penicillin G was active (MIC for 8 strains was 0.5/tg/ml or less ). Exposure to any one of these drugs has been implicated as a predisposing factor for C. spiroforme mediated diarrhoea of rabbits. The greatest variation in MIC was seen for erythromycin (8-fold), penicillin G (8-fold) and tetracycline (16-fold).

The interaction between Porphyromonas gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and IgG light chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of P. gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not alpha 1-antitrypsin nor alpha 2-macroglobulin inhibited the trypsin-like activity in the fluorometric assay.

The Agent of Tyzzer's Disease Is a Clostridium Species. A. JANE DUNCAN, ROBERT J. CARMAN, GARY J. OLSEN, AND KENNETH H. WILSON. From the Department o Medicine and Division ofnfectioos Diseases, Duke University Medical Center, and Veterans Affairs Medical Center, Durham, North Carolina; Techlab Incorporated, VPI Research Park, Blacksburg, Virginia; and the Department of Microbiology, University of Illinois, Urbana, Illinois

A helically coiled, anaerobic, gram-positive sporeforming bacillus, identified as Clostridium spiroforme, was isolated from the cecal contents of all of 27 rabbits with spontaneous diarrhea, at a mean concentration of 10(6.0) spores per g of material. All of these rabbits also had a toxin present in their cecal contents that was neutralized by anti-Clostridium perfringens type E iota toxin, but not by other clostridial antitoxins. In addition, four rabbits with clindamycin-associated colitis were positive for C. spiroforme at a mean concentration of 10(4.5). All of these animals also had iota-like toxin present. Iota-like toxin was not detected in the cecal contents of 72 healthy animals, although C. spiroforme was found in two of these animals at a mean concentration of 10(6.0). C. spiroforme was shown to produce a toxin in vitro that was lethal to mice and caused dermonecrosis in guinea pigs. In all cases, this toxin was neutralized by anti-C. perfringens type E iota toxin.

The dietary carcinogen, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) is mutagenic in the Salmonella/microsomal mutagenicity assay when activated by microsomal enzymes. IQ is found in many cooked foods, notably fried beef and pork. In laboratory rodents IQ is carcinogenic. We showed that mixed and pure cultures of human intestinal anaerobes, notably Eubacterium spp., metabolized IQ to 2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one (HOIQ). Unlike IQ, both the synthetic and bacterially produced HOIQ were direct-acting mutagens, i.e. active without microsomal activation. This new direct-acting mutagen, from the bacterial metabolism of a dietary pyrolysis carcinogen, raises new concerns about the possible role of this class of genotoxins in the etiology of human cancer.

The effects of divalent cations (Ca 2+ , Co 2+ and Zn 2+ ) on the production of iota toxin by Clistridium spiroforme were studied. Toxin production had an absolute requirement for one or more cations in the range 1–5 mM. Using bispecific antisera, we showed that production of both the components of the toxin (i a and i b ) were enhanced by divalent cations added to brain-heart infusion supplemented with peptone and glucose.

Incubation of the heterocyclic cooked food mutagen IQ with mixed human fecal microflora under anaerobic conditions yielded 2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one (2) as the major detectable metabolite.

The enteric diseases of hares, European and cottontail rabbits, which are caused by members of the genus Clostridium are reviewed. Disease caused by C. perfringens Types A and E, C. spiroforme, C. difficile, C. sordellii, C. tympany cuniculi and clostridial enterotoxins are included. Tyzzer's disease also is discussed.

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.

Newly weaned rabbits had diarrhoea only if they were infected with Clostridium spiroforme. In adult rabbits exposure to both clindamycin and C. spiroforme was necessary to induce disease. All diseased animals harboured C. spiroforme and its toxin. Adult rabbits given a course of clindamycin survived when held in a protected environment as did those challenged with C. spiroforme alone. At necropsy none of these apparently healthy animals showed signs of diarrhoea or caecitis. These findings suggest that, in the development of enterotoxaemia, weaning or clindamycin treatment and infection with C. spiroforme are separate events and that disease follows infection with this organism from the environment, as opposed to overgrowth by undetectable levels of C. spiroforme resident in the gut. Our data indicate that C. spiroforme is not a normal component of the rabbit gut flora and that the normal bowel ecology of the adult must be disrupted before C. spiroforme will colonize.

Fifty recent clinical isolates of the B.fragilis group were tested to determine their susceptibility to ampicillin, carbenicillin, mezlocillin, azlocillin and piperacillin using an agar dilution technique. The influence of clavulanic acid on the penicillin susceptibility of these strains was determined by incorporating 2 mg/1 of clavulanic acid into test media. The production of beta-lactamase by the strains was determined by the nitrocefin test and their isoelectric points by focusing on Polyacrylamide gel. Piperacillin was generally the most active of the penicillins tested and carbenicillin the least active with MIC50 values of 8 and 32 respectively. Although mezlocillin was slightly more active than piperacillin against highly resistant strains (MIC 64 mg/1 ampicillin) it was less active against strains with moderate resistance (16–32 mg/1 ampicillin). The majority of resistant strains became at least fivefold more susceptible to ampicillin and carbenicillin in the presence of clavulanic acid, while this effect was less marked for the acylureidopenicillins. In 18% of strains the susceptibility to all five penicillins tested was virtually unaffected by the presence of clavulanic acid.

Five New Zealand White rabbits (Oryctolagus cuniculus) in a rigid barrier rabbit breeding colony developed acute diarrhea 1 week after weaning. Both Clostridium spiroforme and an iota-toxin were isolated from cecal and colon contents of all five rabbits. When pure isolates of C. spiroforme were administered to two normal healthy rabbits, the rabbits developed identical disease and shed both the organism and the iota-toxin. Results of this study suggested that C. spiroforme is an important enteric pathogen of weanling rabbits and the etiology of this diarrhea complex can be rapidly confirmed using four diagnostic criteria.

Seventy seven curved and helically coiled, anaerobic bacilli, which were isolated from the intestinal contents of humans, rabbits and chickens, were the subject of this study. Because they were anaerobic, spore forming, Gram positive rods the isolates were identified as clostridia. Those strains which seemed to have a helically coiled cellular conformation were in fact microcolonies of semicircular cells joined end to end. The strains fell into five groups on the basis of their biochemical characteristics, their susceptibilities to a range of antimicrobial agents, their outer membrane protein and extracellular toxins. Group 1 contained three strains of Clostridium spiroforme, including the type strain, isolated from healthy humans who had not been receiving antibiotics. None of these isolates produced a toxin. Although similar in some respects to the members of Groups 2 and 3, they do in fact represent a distinct group. Groups 2 and 3 share some common extracellular antigens. The former consists of three strains of C. spiroforme isolated from diarrhoeic humans who had received clindamycin, the latter was made up of 59 strains of C. spiroforme from scouring rabbits. All of the rabbit strains, but none of the human isolates, produced a lethal toxin neutralizable with C. perfringens iota antitoxin. Group 4 contained six unnamed strains isolated from healthy chickens. None were toxigenic. They differed in most aspects from the C. spiroforme strains but did share some properties with C. cocleatum, the organism represented in group 5. It is not yet clear whether or not group 2 and 3 should be given species status separate from C. spiroforme. For the time being, however, we are continuing to refer to them as C. spiroforme.

7-hydroxy-IQ is the major product of the bacterial metabolism of IQ, a potent dietary carcinogen. Yet, unlike IQ, hydroxy-IQ is directly active in the salmonella/microsomal mutagenicity assay. Two subjects consumed a meal of fried meats containing IQ but no detectable hydroxy-IQ. Hydroxy-IQ was isolated from the subjects’ faeces collected within 30 h following the fried meat meal; it was absent from the subjects’ faeces before and after the meal. This is the first evidence that hydroxy-IQ is formed in humans. Subsequent concern is therefore raised as to its role in the aetiology of colon cancer. From the faeces of one of the subjects a strain was isolated which conformed to the description of a Eubacterium sp. and was capable of producing hydroxy-IQ.Keywords:-2-amino-3-methyl-3H-imidazo[4,5-ƒ]quinoline;IQ; 2-amino-3,6-3dihydro-3-methyl-7H-imidazo[4,5-ƒ]-quinoline-7-one; Hydroxy-IQ; Intestinal flora; Carcinogens; Faecal Mutagens; Clostridium; Eubacterium moniliforme.

In the late 1970s and early 1980s there were several reports of a fatal diarrhoea of weaned rabbits (1–6). The features of the disease were the demonstration in the caeca of dead animals of a neutralizable iota-like toxin but the absence of detectable Clostridium perfringens Type E, which is known to produce iota toxin. It is now known that the causal organism is C. spiroforme (7–9). In the light of current knowledge, samples from all but one of the earlier outbreaks have been retrospectively analyzed and shown to contain C. spiroforme and its toxin. The condition, previously known as iota enterotoxaemia, can now be better termed C. spiroforme mediated diarrhoea (SMD).

Incubation of the heterocyclic cooked food mutagen 2-amino-3,8-dimethyl-3H-imidazo[4,5-f]quinoxaline (MeIQx) with mixed hulman fecal microflora under anaerobic conditions yielded 2-amino-3,6-dihydro-3,8-dimethylimidazo[4,5-f]-quinoxalin-7-one (7-HOMeIQx, I) as the major metabolite, but with low overall conversion. The metabolite I has been synthesized, it is a direct-acting mutagen, but its isomer is non-mutagenic in the absence of metabolic activation