Clostridium hiranonis sp. nov., a human intestinal bacterium with bile acid 7alpha-dehydroxylating activity.

The Clostridium-like organisms TO-931T and HD-17, isolated from human faeces, have high levels of bile acid 7alpha-dehydroxylating activity. Sequencing of their 16S rDNA demonstrated that they belong to cluster XI of the genus Clostridium and that they represent a new and distinct line of descent. Clostridium bifermentans and Clostridium sordellii in cluster XI also possess bile acid 7alpha-dehydroxylating activity. DNA-DNA hybridization experiments with the isolates, TO-931T and HD-17, and C bifermentans and C. sordellii revealed that the isolates are a single species distinct from C. bifermentans and C sordellii. On the basis of phylogenetic analysis, using 16S rDNA sequences, and DNA-DNA hybridization analysis, it is concluded that strains TO-931T and HD-17 are members of a new species of the genus Clostridium, for which the name Clostridium hiranonis is proposed. The type strain is strain TO-931T (= JCM 10541T = DSM 13275T).

Assignment of Eubacterium sp. VPI 12708 and related strains with high bile acid 7alpha-dehydroxylating activity to Clostridium scindens and proposal of Clostridium hylemonae sp. nov., isolated from human faeces.

Unknown Eubacterium-like organisms VPI 12708 and five strains (Y-1113, I-10, M-18, TH-82 and 36S) had high bile acid 7alpha-dehydroxylating activity; the unknown Clostridium-like organisms TN-271T and TN-272 also had the same activity. Analysis of their 16S rDNA sequences demonstrated that all strains belong to cluster XIVa of the genus Clostridium (Collins et al., 1994). Strain VPI 12708 and five other strains (Y-1113, I-10, M-18, TH-82 and 36S) formed a single cluster and strains TN-271T and TN-272 formed another single cluster. Clostridium scindens JCM 6567T was the most closely related species for two clusters in the phylogenetic tree. Values for DNA-DNA similarities among C. scindens JCM 6567T, strain VPI 12708 and the other five strains were greater than 70%, showing that these micro-organisms were a single species. Therefore, we identified strain VPI 12708 and the five other strains as C. scindens. In addition, DNA-DNA similarities among C. scindens JCM 6567T, strain TN-271T and strain N-272 revealed that strains TN-271T and TN-272 were distinct from C. scindens JCM 6567T. On the basis of phylogenetic analysis and DNA-DNA similarity data, it was concluded that strains TN-271T and TN-272 are members of a new species of the genus Clostridium, for which the name Clostridium hylemonae is proposed. The type strain is strain TN-271T (= JCM 10539T).

Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.

Germ-free mice were orally inoculated with human intestinal 7alpha-dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free-form bile acids, only beta-muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K-5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7alpha-dehydroxylating bacteria, Clostridium species strain TO-931 or Eubacterium species strain 36S.

Isolation of respiratory bacterial pathogens from the throats of healthy infants fed by different methods.

Most bacterial infections are caused by organisms that have already colonized the host. Bacterial attachment to pharyngeal cells and proliferation may be necessary to infect the lower respiratory tract or middle ear. We investigated the incidence of pathogenic bacteria isolated from the throat of healthy infants with different feeding methods. The protecting role of breastmilk is also discussed. The incidence of respiratory bacterial pathogens isolated from the oropharynx of 113 normal infants with different feeding methods was investigated. Group A beta haemolytic Streptococcus, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were selected as respiratory bacterial pathogens. No respiratory bacterial pathogens were detected in breastfed and mixed-fed infants. Haemophilus influenzae and Moraxella catarrhalis were isolated from the oropharynx of formula-fed infants. The incidence of respiratory bacterial pathogens did differ among infants with different feeding methods. These results suggest that breastmilk may inhibit the colonization by respiratory bacterial pathogens of the throat of infants, by enhancing mucosal immunity against respiratory tract infection.

Incidence of Haemophilus influenzae in the throats of healthy infants with different feeding methods.

BACKGROUND: Haemophilus influenzae is the major cause of otitis media and lower respiratory tract infection in childhood. In the presence of human milk, which contains numerous host defense factors, Haemophilus influenzae may be inhibited in attaching to and colonizing pharyngeal cells. We investigated the incidence of H. influenzae in the throats of 162 healthy infants with different feeding methods: 70 breast-fed, 49 mixed-fed and 43 formula-fed infants. METHODS AND RESULTS: Haemophilus influenzae was identified using standard microbiological procedures and the API NH system. The incidence of H. influenzae in breast-fed infants, mixed-fed infants and formula-fed infants was 0, 0 and 7.0% respectively. CONCLUSION: The results suggest that the colonization of H. influenzae in the throat was inhibited by the presence of breast milk.

Assessment of fecal bacteria with bile acid 7 alpha-dehydroxylating activity for the presence of bai-like genes.

Eubacterium sp. strain VPI 12708 has several bile acid-inducible (bai) genes which encode enzymes in the bile acid 7 alpha-dehydroxylation (7 alpha DeOH) pathway. Twelve 7 alpha DeOH-positive intestinal bacterial strains were assayed for 7 alpha DeOH activity, and 13 strains were tested for hybridization with bai genes. Cholic acid 7 alpha DeOH activity varied greatly (> 100-fold) among these strains. Southern blot experiments showed that DNA prepared from 7 of 13 strains hybridized with at least one of the bai genes from Eubacterium sp. strain VPI 12708.

Current status of antimicrobial susceptibility in MRSA isolates typed by coagulase and phage typing in Okinawa.

The incidence of nosocomial infections with methicillin-resistant Staphylococcus aureus is of great concern in Japan and the developed world as a whole. Simple typing techniques like coagulase and phage typing are quick and useful for monitoring and evaluating these organisms. In view of this, the current status of antimicrobial susceptibility in Staphylococcus aureus (S. aureus) isolates in Okinawa typed by coagulase and phage typing was studied. Of 508 isolates, methicillin-resistant S. aureus (MRSA) comprised 54.3% (minimum inhibitory concentration (MIC) > or = 16 micrograms/ml). Coagulase type II and phage group III were the most prevalent, comprising 65.2% and 38%, respectively. These were followed by phage non-typable group and coagulase type III with 36.6% and 12.7%, respectively. Compared to a previous study conducted in 1989, there has been an increase of about 17% in the MRSA isolation rate with a concomitant increase of about 11% in the coagulase type II MRSA isolation rate and a decrease of about 27% in the isolation rate of coagulase type III MRSA. Using a panel of 16 antibiotics, coagulase type II MRSA were resistant to all except Arbekacin and Vancomycin. Arbekacin and Vancomycin were the sole antibiotics to which resistance was not expressed by any of the isolates. With regard to the methicillin-sensitive S. aureus (MSSA), coagulase type III and phase group III were the most prevalent, comprising 25.9% and 32.3%, respectively.

Isolation and characterization of bile acid 7-dehydroxylating bacteria from human feces.

Methods for isolation of fecal 7 alpha-dehydroxylating bacteria are presented. A total of 219 strains were isolated from feces of healthy humans, and their ability to 7-dehydroxylate cholic, chenodeoxycholic, and ursodeoxycholic acids were examined. Of all the isolates, 14 strains were found to be capable of eliminating the hydroxy group at C-7 alpha and/or C-7 beta. All the isolates were strictly anaerobic, Gram-positive rods. Thirteen isolates were non-sporeforming bacteria showing certain saccharolytic properties with the production of acid and gas from dextrose, and were catalase-positive but indole-, lecithinase-, urease- and oxidase-negative. Based on the data available at present, it was concluded that they could be regarded as members of the genus Eubacterium. One strain, however was identified as Clostridium sordellii. The isolated strains capable of 7 alpha-dehydroxylating cholic acid and chenodeoxycholic acid were also able to oxidize the hydroxy group at C-7 alpha. Nine strains (10, 12, 36S, M-2, M-17, M-18, Y-98, Y-1112, and Y-1113) of the 7 alpha-dehydroxylating bacteria were confirmed to have 7 beta-dehydroxylation ability, but five strains (O-51, O-52, O-71, O-72, and Y-67) could not transform ursodeoxycholic acid to lithocholic acid.

Protection of BALB/c mice against methicillin-resistant Staphylococcus aureus infection by intraperitoneal administration of nucleoside-nucleotide mixture.

The effect of intraperitoneally administered nucleic acid components (nucleoside-nucleotide mixture) on the recovery from methicillin-resistant Staphylococcus aureus (MRSA) strain 8985N infection was studied in mice. Two experiments were conducted in which BALB/c mice were fed a nucleotide-free 20% casein diet for 30 days. On the 10th day, the mice were inoculated intravenously with viable MRSA organisms. The mice were intraperitoneally administered nucleoside-nucleotide mixture or saline (control) daily from the onset of the experiment (experiment 1) or from the day of inoculation (experiment 2). The survival rate in the nucleoside-nucleotide group in experiment 1 (70%) was significantly higher than that in the saline group (20%) (p < 0.05). In experiment 2, the survival rate in the nucleoside-nucleotide group (55%) tended to be higher than that in the saline group (36%) without statistically significant difference; furthermore, in the surviving mice, the viable MRSA organisms recovered from the spleen and the kidney were fewer in the former group than in the latter group (p < 0.05). The studies showed that the intraperitoneal administration either before or after the MRSA challenge of the nucleoside-nucleotide mixture was effective for the recovery of the mice from the infection.

The effects of oral RNA and intraperitoneal nucleoside-nucleotide administration on methicillin-resistant Staphylococcus aureus infection in mice.

The effects of oral RNA and intraperitoneal nucleoside-nucleotide mixture administration on methicillin-resistant Staphylococcus aureus (MRSA) strain 8985N infection were studied in mice. BALB/c mice were fed a nucleic acid-free diet or nucleic acid-free diet supplemented with 0.5% or 2.5% ribonucleic acid (RNA) for 30 days. Nucleoside-nucleotide mixture or saline (control) was intraperitoneally administered daily to these rats except for the 2.5% RNA group, which received saline only. On the 10th day of this treatment, the mice were inoculated intravenously with the viable MRSA organisms. Susceptibility to the MRSA was determined by animal survival and recovery of the MRSA from the organs. The survival rates in the three groups that were administered saline were 29%, 35%, and 40% for nucleic acid-free diet, 0.5% RNA, and 2.5% RNA groups, respectively, whereas in the two groups that received the nucleoside-nucleotide mixture the rates were 69% for the nucleic acid-free diet group and 55% for 0.5% RNA group. The susceptibility of the mice to the MRSA challenge was not affected by dietary RNA, which indicates the ineffectiveness of oral RNA. The combined survival rate in the two nucleoside-nucleotide groups (64%) was statistically different (p < .01) from that in the three saline groups (34%). There was a greater reduction in viable organism recovery in the kidney and spleen of the surviving mice that had been administered the nucleoside-nucleotide mixture than in those administered saline.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of intraperitoneally administered nucleoside-nucleotide on the recovery from methicillin-resistant Staphylococcus aureus strain 8985N infection in mice.

The effect of intraperitoneally administered nucleoside-nucleotide on the recovery from methicillin-resistant Staphylococcus aureus (MRSA) strain 8985N infection was studied in mice. Mice fed nucleic acid-free 20% casein diet were administered intraperitoneally with a nucleoside-nucleotide mixture or with saline (control group) daily for 30 days. On the tenth day on this treatment, mice were challenged with the bacteria. The survival rates were 25% and 72% for the control and nucleoside-nucleotide groups, respectively. The recovery of the survived mice from the infection was confirmed by the increment of body weight and the reduction of the bacteria in the organs. The results show the effectiveness of the intraperitoneal administration of the nucleoside-nucleotide mixture for the recovery from the MRSA strain 8985N infection in mice.

[Present status of methicillin-resistant Staphylococcus aureus and susceptibility to antimicrobial agents in 6 hospitals in Okinawa].

During the period from November 1987 to March 1988, 301 strains of S. aureus isolated from clinical specimens were collected from 6 hospitals in Okinawa main island. Thirty seven percent of the strains were resistant to methicillin (MRSA, MIC greater than or equal to 12.5 micrograms/ml). There was a difference in an isolation frequency among the hospitals from 13 to 52%. The strains were isolated from pus, sputum and urine with high frequency. As coagulase typing of MRSA, Type II and III strains predominantly isolated with 54% and 40%, respectively. Among the antimicrobial agents tested, about 90-99% MRSA were resistant to beta-lactam antibiotics and aminoglycosides and no resistant strain was observed to MINO. To IPM/CS and OFLX, 84% and 75% of the strains were sensitive, respectively. MRSA is a hospital strain and may be a causative microorganism for a hospital and opportunistic infection.