Robert J. Carman

Clostridium perfringens toxin genotypes in the feces of healthy North Americans

We investigated the frequency of Clostridium perfringens in the normal fecal flora of healthy North Americans. About half of 43 subjects were colonized with C. perfringens at levels of approximately 10(6)cfu/g feces. Only type A strains were recovered. Spores sometimes outnumbered vegetative cells. Several genotypes were found. Some donors carried two genotypes, some only one. We found no alpha, beta2 or enterotoxin in the stools of any donors. Though some isolates carried toxin genes (e.g. cpe and cpb2) on plasmids, we saw no indication that healthy humans are the reservoir for the chromosomally-borne cpe recovered from cases of C. perfringens food poisoning.

Conversion of IQ, a dietary pyrolysis carcinogen to a direct-acting mutagen by normal intestinal bacteria of humans.

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.

Anaerobic metabolism of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) by human fecal flora

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.

Significance of Clostridium spiroforme in the enteritis-complex of commercial rabbits.

n Abstractn n 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.n n

In vitro susceptibility of rabbit strains of Cloostridium spirofome to antimicrobial agents

Using an agar dilution method we measured the minimum inhibitory concentration (MIC) of 12 antimicrobial agents against 11 strains of iota-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 micrograms/ml or more). Metronidazole was uniformly active against C. spiroforme. With MIC of 8 micrograms/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 micrograms/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).

Isolation, Structure Elucidation, and Synthesis of the Major Anaerobic Bacterial Metabolite of the Dietary Carcinogen 2-Amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (meIQ)

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

Production of iota toxin by Clostridium spiroforme: A requirement for divalent cations

The effects of divalent cations (Ca2+, Co2+ and Zn2+) on the production of iota toxin by Clostridium 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 (ia and ib) were enhanced by divalent cations added to brain-heart infusion supplemented with peptone and glucose.

Taxonomy of Spiral and Curved Clostridia

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.