Clostridium orbiscindens sp. nov., a human intestinal bacterium capable of cleaving the flavonoid C-ring.

Clostridium orbiscindens sp. nov. is an obligate anaerobe that is capable of cleaving the C-3-C-4 bond of the natural anticarcinogen quercetin. The metabolic products, 3,4-dihydroxyphenylacetic acid and presumably phlorglucinol, are not known to possess anticarcinogen properties. This organism was isolated from human feces. On sheep blood agar plates C. orbiscindens forms minute, irregular, convex, gray or white, shiny, smooth, nonhemolytic colonies. It is beta-hemolytic on rabbit blood agar. The motile peritrichous rods are gram variable. Subpolar spores are common. Cultures are resistant to 80 degrees C for 10 min. Capsules are absent. This asaccharolytic organism does not metabolize esculin, urea, meat, gelatin, casein, or nitrate. The G + C content is 56 to 57 mol%. DNA hybridization experiments did not reveal relatedness to phenotypically similar Clostridium strains. Strain 265 (= ATCC 49531) is the type strain.

Evaluation of methods for identification of Campylobacter pyloris infection.

Fifty unselected patients undergoing routine esophagogastroduodenoscopy were evaluated for infection with Campylobacter pyloris (CP). Antral specimens were cultured, and biopsies from the antrum and the body of the stomach were examined histologically. Specimens of antral brushings were analyzed with Gram stain, and urease testing was performed on gastric aspirates, antral brushings, and antral biopsy homogenates. Twenty-seven (54%) patients were CP-positive by silver stain and/or culture of mucosal biopsies. The simplest and fastest diagnostic methods was Gram stain of antral brushing, which was 93% sensitive and 100% specific. CP-negative patients were more likely to have normal histology in antrum and body tissues, while CP-positive patients usually exhibited superficial or chronic gastritis (p less than 0.01). Using ELISA technique, 67% of all patients and 89% of CP-positive patients had serum antibodies against sonicated CP organisms. We conclude that evidence of gastric CP infection is common, is associated with inflammatory changes of the gastric mucosa, is suggested by finding antibodies to CP in serum, and can be accurately and rapidly diagnosed by staining of endoscopically derived cytology and biopsy specimens.

Effects of calcium and bile acid feeding on colon tumors in the rat.

The hypothesis that dietary calcium alters the incidence of colorectal neoplasms was examined in an established model of carcinogenesis. Male Fischer 344 rats (28 days old) were quarantined for 2 weeks. All animals were fed the basal diet (AIN-76) supplemented with cholic acid (0.2%) and/or calcium (1.6%, corresponding to a 3-fold increase above that of the basal diet). N-Methyl-N-nitrosourea (MNU) (2 mg/dose) or saline (control) was given intrarectally to all animals on days 1 and 4 of the experiment. Groups 1-8 were fed the experimental diets concurrently with the first dose of MNU, while groups 9 and 10 were fed the diets 2 weeks prior to MNU (or saline). All animals were sacrificed after 28 weeks. No tumors were observed in the groups given saline (groups 1, 3, 5, 7, and 9). In groups receiving MNU, the addition of cholic acid to the diet (group 4) caused a significant increase in tumors (80% versus 55%), tumors/animal ratio (2.24 versus 0.75), and tumors/tumor-bearing animal ratio (2.80 versus 1.36), group 4 versus group 2, respectively. Increased dietary calcium did not inhibit tumor formation; 68% of animals in groups 6 and 10 had tumors. The combination of dietary cholic acid and calcium (group 8) gave a tumor incidence similar to cholic acid (group 4) alone (72% versus 80%, 2.00 versus 2.24 tumors/animal; 2.77 versus 2.80 tumors/tumor-bearing animal). Most tumors were adenomatous polyps but carcinomas in situ and invasive carcinomas were also present; dietary calcium reduced the number of invasive carcinomas (group 6 versus group 2, P less than 0.04).

C-ring cleavage of flavonoids by human intestinal bacteria.

Four hitherto undescribed Clostridium strains capable of cleaving the C ring of quercetin, kaempferol, and naringenin at C-3-C-4 were isolated from the fecal flora of humans. None of the strains cleaved catechin. C-ring fission occurred when the substrate was either in solution or in suspension. Mixed cultures of flavonoid-hydrolyzing bacteria, flavonoid-cleaving bacteria, and Escherichia coli, which was used to provide the anaerobic environment, rapidly metabolized rutin to 3,4-dihydroxyphenylacetic acid, indicating that the intestinal half-life of the biologically active aglycone is short. The cleaving strains shared many phenotypic characteristics, including their inability to ferment sugars, but they differed sufficiently to indicate that they represent different species.

Urinary excretion of aldosterone metabolite Kelly-M1 in patients with adrenal dysfunction.

Using tetrahydroaldosterone antibody a radioimmunoassay was developed to measure substance Kelly-M1 (K-M1) in human urine. The normal values were lower than observed by Kelly et al. who discovered the catabolite after giving large doses of exogenous aldosterone. While in essential hypertension the excretion of K-M1 was predominantly within the normal range, elevated values were found in most cases of 21-hydroxylase deficiency, both the simple virilizing and salt losing form, primary aldosteronism, renal hypertension and cystinosis. Our findings suggest that K-M1 may be formed from 21-deoxyaldosterone and/or by microbial intervention from aldosterone or its metabolites.

Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans.

Rutin and quercitrin are hydrolysed to quercetin, and robinin is hydrolysed to kaempferol, by faecal flora from healthy subjects. The enzymes required for these hydrolyses, namely alpha-rhamnosidase and beta-galactosidase, were produced by some strains of Bacteroides distasonis; other strains, however, synthesized beta-glucosidase. The last-named enzyme was also elaborated by Bacteroides uniformis and Bacteroides ovatus. All the enzymes were produced constitutively. A cell-free extract of B. distasonis containing beta-glucosidase displayed an enzymic activity of 1 mumol/10 min per 10 mg of protein.

Cofactor requirements of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities in cell extracts of Clostridium scindens.

Two neutral steroid-transforming activities were demonstrated in cell extracts of Clostridium scindens. Steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase were found to be inducible in cells cultured in the presence of cortisol. Both activities required manganese ions and NAD+ or NADH for activity. Cortisol, cortisone and 11-desoxycortisol were substrates as well as inducers of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. 17 alpha-Hydroxyprogesterone was an effective inducer but did not serve as a substrate for either enzyme activity. C. scindens is the first bacterial species of the normal human intestinal flora reported to elaborate inducible steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. The results of cofactor, substrate specificity and induction studies suggest that these two activities may reside in the same enzyme complex.

Bacterial metabolism of natural and synthetic sex hormones undergoing enterohepatic circulation.

Steroids undergoing enterohepatic circulation are exposed to bacterial metabolism particularly by obligate anaerobes which account for 99.99% of the fecal flora. The most common transformation is hydrolysis of conjugated steroids. The glucuronidases are synthesized by Escherichia coli and Bacteroides species. The bacterial catabolism of unconjugated steroids may be considered under several headings: 1. Reduction of ring-A due to clostridia species synthesizing specific enzymes; C. paraputrificum, 3 alpha,5 beta-reductase; C. innocuum, 3 beta,5 beta-reductase; and a new species C.J-1, 3 beta,5 alpha-reductase. 2. Reduction of the delta 5 bond by human fecal flora. The specific strain(s) synthesizing the enzyme have not yet been identified. 3. Reduction of 17-keto estrogens by the above mentioned ring-A reducing clostridia and by Eubacterium lentum. 4. Reduction of 17-keto androstenes by Bacteroides fragilis. 5. Desmolase mediated side chain cleavage at C17-C20 position of 17 alpha-hydroxysteroids by two new species Clostridium scindens and Eubacterium desmolans isolated from human and cat fecal flora respectively and by Clostridium cadavaris isolated from New York City sewage. 6. And 16 alpha- and 21-dehydroxylase by E. lentum a normal inhabitant of the human gut; it is the only organism known to synthesize 16 alpha- or 21-dehydroxylases. Due to the high specificity of the enzymes and the simplicity of extracting the metabolites, biosynthesis of reference compounds and radioimmunoassay reagents is practical and inexpensive. The enzymes can also be used for titration of specific bacterial strains in fecal flora and as markers for bacterial identification in particular for the strains difficult to be defined by regular biochemical reactions.

The 21-acetylation of corticosteroids by Clostridium sporogenes.

A strain of Clostridium sporogenes, an anaerobic bacterium, isolated from sewage in New York City synthesizes two constitutive enzymes with action on steroid molecules: (i) an enzyme capable of selectively acetylating the 21-hydroxyl function of certain steroids and (ii) the corresponding esterase. Under our experimental conditions the enzymes have a strict structural requirement for 3-keto-4-ene and C-20-keto or 20 alpha-hydroxyl group and convert their respective substrates to a mixture of free and acetylated products.

Steroid desmolase synthesis by Eubacterium desmolans and Clostridium cadavaris.

The synthesis of a steroid desmolase was demonstrated in two obligate anaerobes: a new bacterial species, Eubacterium desmolans, isolated from cat fecal flora, and Clostridium cadavaris, recovered from sewage of New York City. The enzyme cleaves the C-17-C-20 bond of corticoids possessing hydroxyl functions at C-17 and C-21. The conversion is quantitative, provided the substrate concentration is less than 100 micrograms/ml and the organisms are in the log phase. The velocity of transformation parallels the bacterial growth curve and in the log phase is higher for E. desmolans than for C. cadavaris. In addition, both organisms synthesize a 20 beta-hydroxysteroid dehydrogenase.

Radioimmunoassay of urinary 21-deoxytetrahydroaldosterone in primary aldosteronism and 21-hydroxylase deficiency.

A radioimmunoassay of 21-deoxytetrahydroaldosterone was developed. Normal daily excretion of the unconjugated metabolite was 1.2 +/- 1.3 micrograms and of the glucuronized metabolite, 11.9 +/- 7 micrograms. The tetrahydroaldosterone/21-deoxytetrahydroaldosterone ratio varied more in patients with primary aldosteronism than in control subjects. Thus, measurements of the urinary excretion of the tetrahydroaldosterone or 21-deoxytetrahydroaldosterone alone did not provide an accurate expression for aldosterone production. Their sum correlated well with the clinical condition, i.e. clear-cut elevation in patients with primary aldosteronism. The diminished tetrahydroaldosterone/21-deoxytetrahydroaldosterone ratio found in patients with 21-hydroxylase deficiency may be attributed to increased bacterial conversion of tetrahydroaldosterone to 21-deoxytetrahydroaldosterone but could also stem from a deficiency implicating zona glomerulosa (aldosterone biosynthesis) regardless of the stage and clinical presentation of the disease.

Performance of the prompt system in identification and antimicrobial susceptibility testing of clinical isolates.

The rapid 3M Prompt inoculation system was compared with the traditional log-phase system for Autoscan identification and antibiotic susceptibility testing (MIC) of 188 recent clinical isolates. The two systems were equally effective for gram-negative rods; the Prompt system was slightly superior for the determination of MICs for gram-positive organisms.

Bacterial formation of aldosterone metabolites.

The experiments described in this paper demonstrate that most of the metabolic alterations of the aldosterone molecule, hitherto attributed to hepatic enzymes, equally well may be carried out by enzymes synthesized by anaerobic bacteria from the human gut. The steroid reductases synthesized by Clostridium paraputrificum, Clostridium J-1, and Clostridium innocuum convert aldosterone to the 3 alpha, 5 beta tetrahydroaldosterone (THA), 3 beta, 5 alpha-THA, and 3 alpha, 5 alpha-THA, respectively. All three enzymes metabolize 5 alpha.dihydroaldosterone to a single compound: 3 beta, 5 alpha-THA. Bifidobacterium adolescentis reduces aldosterone to 20 beta-dihydroaldosterone. In mixed cultures of B. adolescentis and clostridia, the individual enzymes operate independently of each other; however, about half of the aldosterone metabolites are in the free form and half in the acetal form. By appropriate selection of substrate and bacterial strains, therefore, it is possible to biosynthesize not only three of the THA isomers but also the hexahydroisomers in free form as well as in the acetal form.

Mode of action of steroid desmolase and reductases synthesized by Clostridium "scindens" (formerly Clostridium strain 19).

A recently isolated hitherto unknown Clostridium from human feces, designated Clostridium "scindens" (formerly strain 19), synthesizes at least two enzymes active on the side-chain of the steroid molecule and two enzymes active on the hydroxyl groups of the 7-position of bile acids. Steroid desmolase, responsible for side-chain cleavage of corticoids, and 20 alpha-hydroxysteroid dehydrogenase have not been detected in any other bacterial species of the resident colonic flora. Steroid desmolase is Eh-dependent (optimum ca. -130 mV), requires a hydroxy group at C-17, and preferably an alpha-ketol group in the side-chain; an alpha-hydroxy group at C-20 reduces and a beta-hydroxy group at C-20 prevents side-chain cleavage. With suitable substrates, the yield of C-19 steroids is proportional to the bacterial multiplication rate. 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSDH) is also Eh-dependent (optimum ca. -300 mV) and reduces the C-20 keto function to an alpha-hydroxy group, regardless of the presence or absence of a hydroxy group at C-17. 7 alpha-Dehydroxylase metabolizes cholic and chenodeoxycholic acid, while 7 beta-hydroxysteroid dehydrogenase acts upon ursodeoxycholic acid. The latter two enzymes are not specific for C. scindens.

Reduction of 17-keto steroids by anaerobic microorganisms isolated from human fecal flora.

The 17-keto function of phenolic steroids is reduced by the following intestinal bacteria: Eubacterium lentum, Clostridium paraputrificum, Clostridium J-1 and Clostridium innocuum. The 17-keto group of androstenedione is reduced solely by Bacteroides fragilis.

Use of Directigen and acridine orange for rapid identification of human blood culture isolates.

Of 7,871 blood cultures from hospital patients, 22 yielded growth of Streptococcus pneumoniae or Haemophilus influenzae type b. The identities of 19 (86%) of these 22 strains could be verified after 18 to 24 h of incubation by application of the Directigen meningitis test kit to the unheated, uncentrifuged supernatant from the cultures; thus, the turnaround time for these cultures was halved. Growth in 16 (72%) of the Directigen-positive cultures was detected by visual inspection, and that in 3 (14%) of the cultures was detected by acridine orange staining. Growth in the three remaining bottles (14%) was detected by blind subculturing after 18 to 24 h or incubation and, therefore, was delayed by 24 h. The systematic application the acridine orange stain was helpful in 40 (44%) of 91 cases for which macroscopic inspection failed to reveal growth after 24 h of incubation.

Systematic investigation of enrichment media for wild-type Campylobacter jejuni strains.

Of the media examined, thioglycolate broth supplemented with 5% lysed sheep blood, Butzler antibiotic mixture, and 0.1% lauryl sulfate was the most sensitive enrichment medium for recovery of wild-type strains of Campylobacter jejuni from cecal contents of chickens and chicken livers. It allowed the retrieval of 1 CFU as did solid media but permitted the screening of 50-times larger volumes. Double-strength enrichment medium required 5 to 10 CFU for growth. Omission of lauryl sulfate reduced the sensitivity. Replacement of Butzler antibiotic mixture with Blaser antibiotic mixture increased overgrowth and, therefore, decreased retrieval of C. jejuni.

Biosynthesis of androgen from cortisol by a species of Clostridium recovered from human fecal flora.

A hitherto unknown species of Clostridium, provisionally designated strain 19, was isolated from the fecal flora of a healthy human adult. This strain synthesizes a constitutive desmolase that cleaves the side chain of cortisol to form 11 beta-hydroxy-4-androstene-3,17-dione. The enzymatic conversion is best demonstrated in supplemented peptone broth and in prereduced brain-heart infusion broth. The fecal concentration of strain 19 is 10(7)-10(8) cells/g. The strain adapts with difficulty to growth on Mueller-Hinton agar and Columbia agar base; colony formation is enhanced by the addition of 5% sheep blood. The organism is sensitive to penicillin G and resistant to tetracycline, chloramphenicol, clindamycin, and erythromycin.

Inactivation of contraceptive steroid hormones by human intestinal clostridia.

Steroid hormones reduced in ring-A are devoid of hormonal activity. In metabolic experiments we found that human fecal flora reduced the delta 4-3-keto structure of natural progestins to 3 alpha-hydroxy, 5 beta-steroid metabolites (3 alpha,5 beta) and of synthetic progestins to a mixture of 3 alpha,5 beta and 3 beta,5 beta compounds. 3 alpha,5 beta-Reductase was synthesized by Clostridium paraputrificum and had a strong affinity for natural progestins such as progesterone. 3 beta,5 beta-Reductase was synthesized by Clostridium innoculin and had a stronger affinity for synthetic progestins. A third enzyme, 3 beta,5 alpha-reductase, was synthesized by St. Luke's strain 209 (Clostridium species "J-1") but was only observed when pure cultures were used. Ring-A reduction of synthetic progestins was 3 to 10 times slower than that of natural progestins, thus explaining the pharmacological superiority of synthetic progestins over naturally occurring analogs.