In vitro antimicrobial effects of two antihalitosis mouth rinses on oral pathogens and human tongue microbiota.

OBJECTIVES: The aim of the study was to compare the antimicrobial activity of a mouth rinse containing chlorhexidine and cetylpyridinium chloride (MR1) with a stannous fluoride-based mouth rinse (MR2) in vitro. MATERIALS AND METHODS: Samples of the tongues from 10 subjects with and 10 subjects without halitosis were inoculated on blood agar plates. The agar was perforated, and the cylindrical holes were filled either with mouth rinse MR1 or with mouth rinse MR2. After incubation, inhibition zones of the whole tongue microbiota and Fusobacterium nucleatum were measured. In addition, MR1 and MR2 were applied in a short interval killing test (SIKT) on four oral pathogens Porphyromonas gingivalis, Prevotella intermedia, F. nucleatum and Aggregatibacter actinomycetemcomitans. Total viable cell counts were made after two minutes of incubation with increasing concentrations of MR1 and MR2. RESULTS: MR1 showed a significantly higher in vitro antimicrobial activity against the whole tongue microbiota and F. nucleatum than MR2 in both groups of subjects. In the SIK test, MR1 showed a significantly greater killing capacity than MR2. The results show that a mouth rinse with low concentrations of chlorhexidine and 0.05% cetylpyridinium chloride appears to be more effective in inhibiting growth of the human tongue microbiota in vitro than a fluoride/stannous fluoride-containing mouth rinse. CONCLUSION: This in vitro observation supports the use of chlorhexidine and cetylpyridinium chloride in the treatment of oral halitosis.

Effects of yogurt and bifidobacteria supplementation on the colonic microbiota in lactose-intolerant subjects.

AIMS: Colonic metabolism of lactose may play a role in lactose intolerance. We investigated whether a 2-week supplementation of Bifidobacterium longum (in capsules) and a yogurt enriched with Bifidobacterium animalis could modify the composition and metabolic activities of the colonic microbiota in 11 Chinese lactose-intolerant subjects. METHODS AND RESULTS: The numbers of total cells, total bacteria and the Eubacterium rectale/Clostridium coccoides group in faeces as measured with fluorescent in situ hybridization and the faecal beta-galactosidase activity increased significantly during supplementation. The number of Bifidobacterium showed a tendency to increase during and after supplementation. With PCR-denaturing gradient gel electrophoresis, in subjects in which B. animalis and B. longum were not detected before supplementation, both strains were present in faeces during supplementation, but disappeared after supplementation. The degree of lactose digestion in the small intestine and the oro-caecal transit time were not different before and after supplementation, whereas symptom scores after lactose challenge decreased after supplementation. CONCLUSIONS: The results suggest that supplementation modifies the amount and metabolic activities of the colonic microbiota and alleviates symptoms in lactose-intolerant subjects. The changes in the colonic microbiota might be among the factors modified by the supplementation which lead to the alleviation of lactose intolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence for the possibility of managing lactose intolerance with dietary lactose (yogurt) and probiotics via modulating the colonic microbiota.

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics.

The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus. The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis, are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative "secretomics" approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the "gadgets" that S. aureus needs to conquer these well-protected niches.

A Desulfitobacterium strain isolated from human feces that does not dechlorinate chloroethenes or chlorophenols.

An anaerobic bacterium, strain DP7, was isolated from human feces in mineral medium with formate and 0.02% yeast extract as energy and carbon source. This rod-shaped motile bacterium used pyruvate, lactate, formate, hydrogen, butyrate, and ethanol as electron donor for sulfite reduction. Other electron acceptors such as thiosulfate, nitrate and fumarate stimulated growth in the presence of 0.02% yeast extract and formate. Acetate was the only product during fermentative growth on pyruvate. Six mol of pyruvate were fermented to 7 mol of acetate. 13C-NMR labeling experiments showed homoacetogenic 13C-CO2 incorporation into acetate. The pH and temperature optimum of fermentative growth on pyruvate was 7.4 and 37 degrees C, respectively. The growth rate under these conditions was approximately 0.10 h(-1). Strain DP7 was identified as a new strain of Desulfitobacterium frappieri on the basis of 16S rRNA sequence analysis (99% similarity) and DNA-DNA hybridization (reassociation value of 83%) with Desulfitobacterium frappieri TCE1. In contrast to described Desulfitobacterium strains, the newly isolated strain has not been isolated from a polluted environment and did not use chloroethenes or chlorophenols as electron acceptor.

Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria.

Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared from fecal samples of three healthy volunteers. To introduce variation between the samples, they were incubated for 48 h in batch culture (anaerobic) fermenters at 37 degrees C, and pure cultures of Bifidobacterium infantis, Clostridium perfringens, or Lactobacillus acidophilus were added. Samples were taken from the fermenters at different times. Total anaerobes, bifidobacteria, bacteroides, clostridia, and lactobacilli were enumerated by both plating and FISH. The results showed that plate counts of total anaerobes, bifidobacteria, lactobacilli and bacteroides were approximately ten-fold lower than the corresponding FISH counts. Numbers of clostridia were higher using the plating method, probably because the clostridia probe used in FISH analyses was designed to only detect part of the genus Clostridium. The introduced variation in the methods could be detected by both methods and was comparable.

Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods.

BACKGROUND: An obvious difference between breast-fed and formula-fed newborn infants is the development of the intestinal flora, considered to be of importance for protection against harmful micro-organisms and for the maturation of the intestinal immune system. In this study, novel molecular identification methods were used to verify the data obtained by traditional culture methods and to validate the culture independent fluorescent in situ hybridization (FISH) technique. METHODS: From each of six breast-fed and six formula-fed newborn infants, six fecal samples were obtained during the first 20 days of life. The microbial compositions of the samples were analyzed by culturing on specific media and by FISH, by using specific 16S rRNA-targeted oligonucleotide probes. The colonies growing on the media were identified by random amplified polymorphic DNA pattern analysis and by polymerase chain reaction amplification and subsequent analysis of the 16S rRNA gene. RESULTS: Molecular identification of the colonies showed that the selective media are insufficiently selective and unsuitable for quantitative analyses. Qualitative information from the culturing results combined with the data obtained by the FISH technique revealed initial colonization in all infants of a complex (adult-like) flora. After this initial colonization, a selection of bacterial strains began in all infants, in which Bifidobacterium strains played an important role. In all breast-fed infants, bifidobacteria become dominant, whereas in most formula-fed infants similar amounts of Bacteroides and bifidobacteria (approximately 40%) were found. The minor components of the fecal samples from breast-fed infants were mainly lactobacilli and streptococci; samples from formula-fed infants often contained staphylococci, Escherichia coli, and clostridia. CONCLUSIONS: This study confirms the differences in development of intestinal flora between breast-fed and formula-fed infants. The results obtained from the FISH technique were consistent. Although the repertoire of probes for this study was not yet complete, the FISH technique will probably become the method of reference for future studies designed to develop breast-fed-like intestinal flora in formula-fed infants.

Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes.

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 x 10(10) cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 x 10(10) cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 x 10(7) to 7 x 10(8) per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.

16S ribosomal RNA-targeted oligonucleotide probes for monitoring of intestinal tract bacteria.

BACKGROUND: The composition of a sample of faecal bacteria can be determined by culturing different dilutions on specific media. However, not all bacteria can be cultured and media are not always specific. With a culture-independent approach a more accurate picture of the composition of the intestinal flora may be obtained. METHODS: Fluorescently labelled oligonucleotide probes targeted at 16S ribosomal RNA sequences specific for a bacterial genus were designed and applied for fluorescence in situ hybridization (FISH) of bacteria in human faecal samples. RESULTS: The mean number of Bifidobacterium spp. and the total number of anaerobic bacteria per gram of faeces were determined by culturing and with the probe technique. Although in both cases the number of Bifidobacterium spp. was about the same, 2.38 x 10(9) and 2.45 x 10(9), it was found that the contribution of Bifidobacterium spp. to the total composition is overestimated due to the lower number of total anaerobic bacteria estimated by culturing. CONCLUSION: Genus-specific or group-specific fluorescent 16S rRNA probes may become an invaluable tool in gut ecology studies.

Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples.

Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.