Tsutomu Ushijima

The inhibition of free radical generation by human neutrophils through the synergistic effects of metronidazole with palmitoleic acid: a possible mechanism of action of metronidazole in rosacea and acne

SummaryMetronidazole is clinically effective in treating not only rosacea but also acne inflammation. Yet it is generally considered not to be very effective in inhibiting the growth of anaerobic Propionibacterium acnes. We report here our investigation into the synergistic effects of metronidazole and palmitoleic acid on the anaerobic growth of P. acnes as well as on human neutrophil functions, including the generation of reactive oxygen species (ROS). Both metronidazole and palmitoleic acid, when used alone, only slightly inhibited the growth of P. acnes, and no significant decrease in human neutrophil functions, including the generation of ROS, was observed. But metronidazole used in the presence of palmitoleic acid markedly inhibited the anaerobic growth of P. acnes and decreased ROS generation by neutrophils. However, ROS generated in the xanthine-xanthine oxidase system were not affected. Metronidazole was shown to be clinically effective by decreasing neutrophil-generated ROS at the sites of inflammation with the aid of palmitoleic acid, which is generally present in human skin. By inhibiting oxidative tissue injury under in vivo conditions, treatment with metronidazole results in remarkable improvement of rosacea and acne.

Selected faecal bacteria and nutrients essential for antagonism of Salmonella typhimurium in anaerobic continuous flow cultures

As few as five of the species of bacteria commonly found in human faeces--Escherichia coli, Enterobacter aerogenes, Enterococcus faecalis, Bacteroides ovatus and Fusobacterium varium--when grown together in anaerobic continuous flow cultures exerted antagonistic effects on Salmonella typhimurium as great as those given by mixed bacteria from extracts of human faeces. In a single culture, the population of S. typhimurium was c. 10(8) cfu/ml but in mixed cultures with the five antagonistic bacteria or mixed faecal bacteria it was reduced to c. 10(3) cfu/ml. Antagonism appeared to be the result of competition for the growth limiting amino acids, arginine, serine, threonine and aspartic acid. Optimal manifestation of antagonism required the presence of carbon sources fermentable only by antagonistic bacteria, such as lactose 0.1%, w/v, sucrose 0.1% (w/v) and starch 0.2-0.3% w/v. These carbohydrates promoted the growth of the antagonistic bacteria, particularly E. coli and B. ovatus. However, an increase in concentration by several fold of any one of four growth-limiting amino acids in the medium diminished the antagonistic effects and the population of S. typhimurium rose 10(2)-10(3)-fold.

Crystallization and properties of aromatic amine dehydrogenase from Pseudomonas sp

An amine dehydrogenase was purified to homogeneity from an extract of a bacterium of the genus Pseudomonas grown in a medium containing beta-phenylethylamine as a sole carbon source and obtained in a crystalline form with about 100-fold purification. The purified enzyme catalyzed the oxidative deamination of various aromatic amines as well as some aliphatic amines to a lesser extent. An artificial electron acceptor such as phenazine methosulfate was required for the catalysis. The molecular weight determined by sedimentation equilibrium was 103,000 and the molecule seemed to be composed of two pairs of two nonidentical subunits (Mr 46,000 and 8000). The enzyme had a dull yellow-green color with an absorption maximum at 445 nm and this chromophore appeared to be involved in the catalytic action of the enzyme.

Clostridium ramosum, an IgA Protease-Producing Species and Its Ecology in the Human Intestinal Tract

A bacterial strain isolated from feces of a patient with ulcerative colitis, which had been shown to produce a novel immunoglobulin A (IgA) protease (cleaving both the human IgA1 subclass and IgA2 subclass of A2m(1) allotype) extracellularly, was identified as Clostridium ramosum. By using a selective medium (proprion‐ate‐rifampicin‐gentamicin‐colimycin‐polymyxin medium) devised for C. ramosum, analysis of the population level of this organism was performed to determine its ecology in the human intestinal tract. C. ramosum was isolated in 20 of 25 fecal samples (80%) from patients with inflammatory bowel disease (I.B.D.) and in 112 of 135 samples (83%) from patients without I.B.D. (control group). C. ramosum was also isolated from 6 of 11 biopsy samples (55%) of the inflamed rectal mucosa from patients with ulcerative colitis and from five of 15 samples (33%) from the intact mucosa of the control group. The population levels of C. ramosum in most of the biopsy samples ranged from 2.3 to 5.0 log10 per gram. The IgA protease‐positive C. ramosum was found in only four of 135 fecal samples (3%) and one of 15 biopsy samples (6.7%) from the control group. These results indicate that IgA protease‐positive C. ramosum is not likely to play a role in the induction of I.B.D., unless the organism is first isolated from the patient with I.B.D.

A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group

A new selective medium, Bacteroides fragilis ammonium‐sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram‐positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.

Effects of Subminimal Inhibitory Concentrations of Erythromycin, Tetracycline, Clindamycin, and Minocycline on the Neutrophil Chemotactic Factor Production in Propionibacterium acnes Biotypes 1–5

Biotypes 1–5 Propionibacterium acnes (P. acnes) strains were grown in the presence of 1/10 minimal inhibitory concentrations (sub‐MIC) of erythromycin (EM), tetracycline (TC), clindamycin (CLDM), or minocycline (MINO) and their culture filtrates were assayed for human neutrophil chemotactic activity using Boyden chamber methods.

Selective and Differential Media for Isolation and Tentative Identification of Each Species of Pityrosporum Residing on Normal or Diseased Human Skin

Selective and differential media were designed for each species of Pityrosporum; P. pachydermatis, P. ovale, and P. orbiculare in order to make feasible a quantitative cultivation.

Potent Antagonism of Escherichia Coli, Bacteroides Ovatus, Fusobacterium Varium, and Enterococcus Faecalis, Alone or in Combination, for Enteropathogens in Anaerobic Continuous Flow Cultures

Interactions between representative strains of four predominant resident bacteria of the human colon, Escherichia coli, Enterococcus faecalis, Bacteroides ovatus, and Fusobacterium varium, and strains of seven enteropathogens, Yersinia enterocolitica, Shigella flexneri, Salmonella typhimurium, Vibrio parahaemolyticus, V. cholerae serogroup non O1, Staphylococcus aureus, and Clostridium perfringens, were examined in studies with an anaerobic continuous flow culture system and medium resembling the content of the mouse caecum (MCM). Potent unilateral antagonism attributable to synergic activities of the resident bacteria against the enteropathogens was evident. The four resident bacteria persisted at levels of c. 10(6) cfu/ml or more in single and in any mixed cultures of the resident species. The seven enteropathogens also persisted in single cultures. In contrast, Y. enterocolitica was excluded in several days in mixed cultures with each of the four resident bacteria. Sh. Flexneri and Staph. aureus were excluded in the presence of E. coli alone. C. perfringens, V. parahaemolyticus and V. cholerae serogroup non O1 were excluded in the presence of E. coli with B. ovatus and, in some cases, with additional species. S. typhimurium was the most resistant; only c. 10(2)-fold reduction of the population level was observed in mixed culture with all four of the resident species. When the amounts of some components in the medium, such as peptone and yeast extract, were increased, C. perfringens grew and persisted even in the presence of the four resident bacteria. Sh. flexneri, in contrast decreased steadily, even in enriched media.

Mode of protection of mice against herpes simplex virus type 2 infection by Propionibacterium

We compared various strains of Propionibacterium with regard to protection of young adult mice against lethal infection with herpes simplex virus type 2 (HSV‐2). Propionibacterium acnes, P. granulosum, and P. avidum were protective, while P. acidi‐propionici and P. lymphophilum were ineffective. The protective effect proved to be in the cell wall fraction. Attempts were made to elucidate possible mechanisms of the protection using both effective and ineffective strains. The results strongly suggest that induction of interferon rather than activation of macrophages and natural killer cells by Propionibacterium pretreatment plays a crucial role, directly or indirectly, in protection against infection by herpes simplex virus. Propionibacterium only moderately protected newborn mice against HSV‐2 infection.

Changes in hepatic superoxide dismutase and xanthine oxidase activity in mice infected with Salmonella typhimurium and Pseudomonas aeruginosa.

Liver xanthine oxidase (XOD) and superoxide dismutase (SOD) activities were compared in mice during Salmonella typhimurium and Pseudomonas aeruginosa infections. We observed that XOD activity rose but SOD activity fell for the first 11 days after infection with smooth type S. typhimurium, coinciding with the period of bacterial growth in the liver. Rough type S. typhimurium did not establish an infection and mice inoculated with this strain showed no variation in enzyme activities. P. aeruginosa infection was mild but stimulated both XOD and SOD activities.