Biomedical subjects
Publications and source records attributed to N Masuda.
The addition of flavins to the growth medium specifically enhanced the 7alpha-dehydroxylation of bile acids by anaerobically growing cultures of a Eubacterium lentum-like intestinal anaerobe, strain c-25, without an increase in cell growth. The order of the enhancement of the reaction was flavin adenine dinucleotide > flavin mononucleotide >> riboflavin.
Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.
Human isolates of Actinomyces viscosus and Actinomyces naeslundii have been divided into six clusters in a numerical taxonomy study. Surface fibrils of strains representing these clusters were isolated and purified. Chemical analyses revealed that the major component of all fibrils was protein and that although differences in percentages of specific amino acid residues were found, the relative proportions of basic, acidic, polar uncharged, and nonpolar amino acids were rather similar among clusters. All of the fibrils except those from strain B236 (cluster 2) either failed to migrate or penetrated only slightly into gels during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after boiling, reduction, or alkylation. Immunological studies by electron microscopic examination of fibril-antibody immunocomplexes, whole bacterial cell agglutination, inhibition of hemagglutination, and immunofluorescence by using antifibril antisera and antibodies demonstrated that strains of typical A. naeslundii (cluster 5) have a specific fibril-associated antigen(s) distinct from those of strains of other clusters. Cross-reactions for atypical A. naeslundii (cluster 3) were few. The fibrils from A. viscosus clusters 1, 2, 4, and 6 demonstrated several cross-reactions. By absorbing antifibril antibodies with cross-reactive strains it was possible to obtain cluster-specific antibodies, as determined by whole cell agglutination, only for cluster 5. Absorbed antifibril antisera for both A. naeslundii clusters 3 and 5 were specific by indirect immunofluorescence, whereas anti-cluster 1 fibril antisera cross-reacted only with other A. viscosus cluster representatives. Purification of Actinomyces fibrils by methods used for appendages of other species yields preparations containing common antigens among taxonomic groups. However, absorbing antifibril antisera, gamma globulin, or both has promise for producing cluster-specific reagents useful in identification.
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Cefroxadine (CXD) is an orally administered synthesized cephalosporin antibiotic developed by Ciba-Geigy Limited (Switzerland) in 1972. We have studied the clinical effectiveness of this drug in a total of 45 cases of various types of infections in the dentistry and the oral surgery. The studies resulted in showing 18 markedly effective cases, 19 effective cases, 5 slightly effective cases, 1 ineffective case, and 2 unknown cases showing an effective rate of 82.2%. Side effects manifested in 2 cases, of which 1 case was considered to be attributable to CXD, and the occurrence frequency of side effects was as low as 2.2%. In bacteriological test, there were many cases of mixed infections by Gram-positive and Gram-negative bacteria, and these infections were those which are observed in high frequency in dentistry and oral surgery infections. As a result of an overall evaluation of CXD clinical effects, the drug considered to be an antibiotic which is highly useful in dentistry and oral surgery.
Peptostreptococcus productus strain b-52 (a human fecal isolate) and Eubacterium aerofaciens ATCC 25986 were found to contain NADP-dependent 7 beta-hydroxysteriod dehydrogenase activity. The enzyme was synthesized constitutively by both organisms, and the enzyme yields were suppressed by the addition of 0.5 mM 7 beta-hydroxy bile acid to the growth medium. Purification of the enzyme by chromatography resulted in preparations with 3.5 (P. productus b-52, on Sephadex G-200) and 1.8 (E. aerofaciens, on Bio-Gel A-1.5 M) times the activity of the crude cell extracts. A pH optimum of 9.8 and a molecular weight of approximately 53,000 were shown for the enzyme of strain b-52, and an optimum pH at 10.5 and a molecular weight of 45,000 was shown for that from strain ATCC 25986. Kinetic studies revealed that both enzyme preparations oxidized the 7 beta-hydroxy group in unconjugated and conjugated bile acids, a lower Km value being demonstrated with free bile acid than with glycine and taurine conjugates. No measureable activity against 3 alpha-, 7 alpha-, or 12 alpha-hydroxy groups was detected in either enzyme preparation. When tested with strain ATCC 25986, little 7 beta-hydroxy-steroid dehydrogenase activity was detected in cells grown in the presence of glucose in excess. The enzyme from strain b-52 was found to be heat labile (90% inactivation at 50 degrees C for 3 min) and highly sensitive to sulfhydryl inhibitors.
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The 7 alpha-dehydroxylation of chenodeoxycholic acid (CDCA) and cholic acid (CA) by a Eubacterium lentum-like intestinal anaerobe was specifically enhanced by the bacteroides present in mixed cultures and also by the addition to the growth medium of cell extracts from the bacteroides. The 7 alpha-dehydroxylating organism also possessed 7 alpha-hydroxysteroid dehydrogenase activity, and, in collaboration with a 7 beta-dehydrogenating organism, converted ursodeoxycholic acid (UDCA) into CDCA. Large quantities of lithocholic acids were produced from UDCA as well as CDCA in in vitro cocultures of these three kinds of microorganisms.
Deconjugation of bile salts by four strains of Bacteroides and four strains of Clostridium was studied by use of resting cells and cell-free culture supernatants. Bacteroids strains yielded active cells but showed relatively low bile salt hydrolase (BSH) activity in the culture supernatants while the reverse was the case for the spore-forming clostridial strains. BSH was formed constitutively and was oxygen insensitive. The optimum pH was between 4.5 and 5.0. Marked substrate specificity was found in two strains, one Clostridium and one Bacteroides, which showed restricted activity against taurine conjugates. Bacteroides in general attacked the taurine conjugates of dihydroxy bile acids more readily than the trihydroxy taurine conjugates. Deconjugated bile acid moieties were further modified by some resting cells, depending on the bacterial strain while no enzymatic activity other than that of BSH was found in the culture supernatants. Cells of B. fragilis 2536 performed 7 alpha-dehydrogenation when the pH of the medium allowed the reaction, and this oxidative process was markedly enhanced in the presence of an abundant supply of oxygen as a terminal electron acceptor. C. perfringens PB 6K produced the 3- keto product in addition to the 3 beta-hydroxy derivative of the liberated bile acids and the formation of the latter derivative seemed to take place without preliminary deconjugation.
Microbiol transformation of cholic acid and chenodeoxycholic acid by anaerobic mixed cultures of human fecal microorganisms was investigated, and the results were examined in relation to the bile acid transforming activities of 75 bacterial strains isolated from the same fecal cultures. The reactions involved in the mixed cultures were dehydrogenation and dehydroxylation of the 7 alpha-hydroxy group in both primary bile acids and epimerization of the 3 alpha-hydroxy group in all metabolic bile acids. Extensive epimerization of the 7 alpha-hydroxy group of chenodeoxycholic acid yielding ursodeoxycholic acid was also demonstrated by certain fecal samples. 7 alpha-Dehydrogenase activity was widespread among the fecal isolates (88% of 16 facultative anaerobes and 51% of 59 obligate anaerobes), and 7 alpha-dehydroxylase activity was revealed in one of the isolates, and unidentified gram-positive nonsporeforming anaerobic bacterium. 3 alpha-Epimerization was effected by seven strains assigned to Eubacterium lentum, which were also active for 3 alpha- and 7 alpha-dehydrogenations. No microorganism accounting for 7 alpha-epimerization was recovered among the isolates. Splitting of conjugated bile acid was demonstrated by the majority of obligate anaerobes but the activity was rare among facultative anaerobes.
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Thirteen anaerobic bacteria capable of performing the 7 alpha-dehydroxylation of both cholic acid and chenodeoxycholic acid were isolated from human feces and also from sewage. Ten organisms from heat-treated samples were species of Clostridium identical or closely related to the Clostridium bifermentans-C. sordellii group and consisted of four strains elaborating 7 alpha-dehydroxylase alone and six strains capable of catalyzing both 7 alpha-dehydrogenation and 7 alpha-dehydroxylation. The remaining three organisms, recovered from fresh human feces, were gram-positive, nonflagellated, nonsporeforming, anaerobic rods and comprised two distinct species. Strain HD-17, still unidentified, had both activities, but was unique in that it exclusively 7 alpha-dehydroxylated cholic acid while biotransforming chenodeoxycholic acid, preferably though 7 alpha-dehydrogenation. Two unclassified strains, b-8 and c-25, metabolized both acids though 7 alpha-dehydroxylation and 7 alpha-dehydrogenation. Except for strains b-8 and c-25, all of th 7 alpha-dehydroxylating bacteria split the conjugated bile acid series, and hydrolases were detected in cell-free filtrates of early stationary-phase broth cultures.
Thirty-five strains of Clostridium perfringens were examined for their ability to transform bile acids, both in growing cultures and by washed whole cells. All of the strains oxidized the 3 alpha-hydroxy group to an oxo group, and all except three converted the same alpha-hydroxy group into a beta-configuration. The oxidative 3 alpha-dehydrogenation was barely detectable under anaerobic cultural conditions but was clearly demonstrated in an aerated system using washed whole cells, with a pH optimum between 7.0 and 9.0. The epimerizing reaction amounting to 10 to 20% conversion was observed in anaerobic cultures and also with resting cells, irrespective of oxygen supply. Both reactions were carried out with seven conventional 3 alpha-hydroxy bile acids, thus producing a series of 3-oxo and 3 beta-hydroxy derivatives that could be examined for gas-liquid chromatographic and mass spectrometric behavior. No evidence for the occurrence of 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase activities among the test strains was found. A highly potent deconjugating hydrolase was elaborated by all of the strains.
A group of fecal isolates identified as Eubacterium lentum elaborated 3 alpha-, 7 alpha-, and 12 alpha-dehydrogenases and also an epimerizing enzyme(s) for the 3 alpha-hydroxy group. The activities of the enzymes, however, were variably manifested according to the kind of bile acid substrate and the oxygen tension under which the reaction occurred.
Fibrils of Actinomyces viscosus WVU627 (numerical taxonomy cluster 1) were obtained by homogenization and purified by ultrafiltration, ammonium sulfate precipitations, gel filtration, and ion-exchange chromatography. Electron microscopy and resolution of a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis attested to the purity of the preparation. Purified fibrils were composed mainly of protein; small quantities of carbohydrate and phosphorus were detected. Immunoelectrophoresis revealed only a single precipitable antigen, which migrated slightly toward the anode, in reactions between purified fibrils and antiserum raised against either whole bacterial cells or the purified fibrils themselves. Immunoelectron microscopy with ferritin-conjugated antifibril antibody hemagglutination inhibition, and bacterial agglutination tests demonstrated that fibrils of Actinomyces viscosus cluster 1 strains shared some common antigens with clusters 2, 3, 4 and 6, but did not cross-react with typical Actinomyces naeslundii of cluster 5. Stability tests revealed that after heat or alkali treatment, the fibrils lost their antigenicity and disappeared from electron micrographs. They were affected less by sodium dodecyl sulfate, sonic, or acid treatments.
An unidentified gram-positive anaeroic organism capable of dehydrogenating the 7 beta-hydroxy group of ursodeoxycholic acid was recovered from human feces. By combined action of this organism with the ordinary 7 alpha-dehydrogenating bacteria, chenodeoxycholic acid and cholic acid alike were readily converted into their respective 7 beta-epimers and the reverse reactions were also carried out. The estimated levels of these 7 alpha- and 7 beta-dehydrogenating organisms among the intestinal microflora give a satisfactory explanation for the frequent appearance of the 7 beta-hydroxylated bile acids in vivo.
Nine fecal samples from four healthy subjects were examined for their ability to transform chenodeoxy-cholic acid (CDCA) and ursodeoxycholic acid (UDCA) in in vitro anaerobic broth cultures. Seven samples converted CDCA and UDCA into each other (more than 50% of CDCA was converted into UDCA while 10% or less of UDCA was converted into CDCA), and produced 7-keto-lithocholic acid and lithocholic acid equally from both acids. No alteration of the 7 beta-hydroxy group of UDCA was demonstrated by two fecal samples that failed to perform mutual 7-epimerization, suggesting the conversion of UDCA into lithocholic acid via CDCA. The 3 alpha-hydroxy groups of these substrate and metabolite bile acids were invariably partially epimerized to 3 beta-hydroxy groups by all the fecal samples. Evidence is presented for the prevalence of these 7- and 3-epimerizing organisms among the human intestinal flora.
Streptococcus mutans were detected in the feces from 10 of 29 caries-active patients, aged 4 to 9 years. The percentage of S. mutans to the total counts of facultatively anaerobic streptococci on mitis salivarius agar (Difco Laboratories) varied from 0 to 72.5%. S. mutans were then isolated from dental plaque of sound teeth and carious dentin of the 10 subjects known to harbor S. mutans in the feces. The frequency distribution of various serotypes of these dental and fecal isolates of S. mutans was compared by the immunodiffusion technique. Of the total 1,047 isolates (290 isolates from feces, 289 from dental plaque, and 468 from carious dentin), type c isolates were most prevalent (ca. 66%). Serotype d, e, f, and g isolates were also found but in far lower frequencies. Plural serotypes of S. mutans were occasionally found in dental and fecal samples of a single subject. For two subjects, relatively rare serotypes of S. mutans in the population examined, serotype e, f, or g, were predominantly found in their fecal and dental samples and those of their siblings and mother, suggesting an intrafamilial transmission of S. mutans.