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PubMed2002

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries.

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.

Animals↗
PubMed2002

Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods.

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture-based method. Direct counts ranged from 1.9 X 10" to 4.0 X 10" cells/g (wet weight), and plate counts totaled 6.6 X 10(10) to 1.2 X 10(11) CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.

Adult↗
PubMed2003

Terminal restriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities.

Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize and compare human fecal microbiota among individuals. T-RFLP patterns of fecal 16S ribosomal DNA (rDNA) PCR products from three adults revealed host-specific bacterial communities and were in good agreement with those reported in our previous study. In addition, we applied T-RFLP analysis for the analysis of complex bifidobacterial communities in human fecal samples. The developed method based on Bifidobacterium genus-specific PCR and T-RFLP could identify more than one bifidobacterial species. T-RFLP patterns of Bifidobacterium genus-specific PCR products from the fecal samples were host-specific as well as those of fecal 16S rDNA PCR products. These results were confirmed by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the genus Bifidobacterium and Bifidobacterium species- and group-specific PCR. Our study demonstrates that T-RFLP analysis is useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals, and that the applied method is useful for rapid and sensitive analysis of bifidobacterial community.

Adult↗
PubMed1989

Inhibition by antibiotics of the bacterial response to long-term starvation of Salmonella typhimurium and the colon microbiota of mice.

The number of viable cells of two strains of Salmonella typhimurium and the number of viable cells and the cell size of the colon microbiota of mice were examined during non-growing conditions after exposure to antibiotics with known modes of action. Salmonella typhimurium starved for 1, 2, 4, 5, 12 and 20 d in a phosphate buffer saline solution and subsequently exposed for 2 and 6 h showed the following characteristics. The protein synthesis inhibitors gentamicin and tetracycline, the RNA synthesis inhibitor rifampicin and the membrane potential inhibitor polymyxin all impaired survival of starved cells. The reduction in the number of viable cells caused by the addition of gentamicin, rifampicin and polymyxin was generally more pronounced with extended exposure to energy and nutrient deprivation. Both 2- and 6-h exposure of tetracycline, however, had diminishing inhibitory effects after 20 d compared with 5 d of starvation. Control experiments to verify non-growing conditions in the starvation regime showed that DNA and cell wall synthesis inhibitors had no inhibitory effect after 24-h starvation. The rough mutant strain displayed a lower sensitivity to a hydrophobic rather than a hydrophilic inhibitor as compared to the smooth wild-type strain. The cell size reduction but not viability was partly prevented by protein synthesis inhibitors as seen for both in vivo and in vitro colon microbiota studies.

Animals↗
PubMed2006

Commensal bacilli inhibitory to mastitis pathogens isolated from the udder microbiota of healthy cows.

AIMS: To isolate from the microbiota of the healthy cow udder commensal bacteria having antimicrobial activity against bovine mastitis pathogens, with a long-term view to their potential application as antimastitis probiotics. METHODS AND RESULTS: Bacterial isolates from four healthy cow udders were tested for inhibitory activity against three Gram-positive indicator bacteria. This led to the selection of nine broadly inhibitory strains. All were of the Bacillus genus and their antimicrobial activities, which appeared heterogeneous on the basis of their antibacterial spectra and heat susceptibilities, enabled grouping of the inhibitory bacilli into six different inhibitory profiles. All displayed strong in vitro activity against Gram-positive mastitis pathogens. Inhibitory bacilli were recovered from each of the 11 udder samples collected over 7 months from one of these cows and the isolates included representatives of all six inhibitory profiles. CONCLUSIONS: Bacilli present in the udder microbiota of healthy cows can produce a variety of broadly active inhibitors of Gram-positive bacteria, including potential mastitis pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Inhibitor-producing strains of commensal Bacillus species have been identified, which may have the potential for use as possible antimastitis probiotics.

Animals↗
PubMed1989

The capacity of subgingival microbiotas to produce volatile sulfur compounds in human serum.

Hydrogen sulfide is formed by the subgingival microbiotas of periodontal pockets. The capacity of these microbiotas to form various volatile sulfur compounds in human serum was studied. Bacterial samples from nine deep periodontal pockets were incubated for 7 days in human serum and the amounts of volatile sulfur compounds and the degradation of serum proteins were determined. Hydrogen sulfide was the predominant volatile sulfur compound, but also methyl mercaptan was formed in significant amounts. Only traces of dimethyl sulfide and dimethyl disulfide were detected. There was an extensive degradation of the serum proteins. In most of the reaction mixtures hydrogen sulfide reached highly toxic levels.

Bacteria↗
PubMed1989

The predominant cultivable microbiota of crevicular epithelial cells.

The purpose of the present investigation was to compare the composition of the predominant cultivable microbiota associated with gingival crevicular epithelial cells with that of the unattached microbiota recovered from the same site. Samples were taken from 2 diseased sites from 8 periodontal patients, by scraping the epithelial lining of the pocket with a curette. The epithelial cells were separated from the unattached subgingival bacteria by centrifugation in a reduced 50% Percoll density gradient. Epithelial cells formed a band at the top of the gradient and were removed separately from the unattached bacteria located at the base. Each layer was dispersed, diluted and plated on Trypticase soy agar with 5% sheep blood and 50 isolates were characterized from each sample. The microorganisms associated with the epithelial layer harbored 5- to 20-fold higher mean percentages of Bacteroides gingivalis, Bacteroides intermedius and Peptostreptococcus micros. The layer of unattached organisms exhibited 4- to 10-fold higher mean percentages of Streptococcus uberis, Capnocytophaga ochracea, Eikenella corrodens and Veillonella parvula.

Colony Count, Microbial↗
PubMed1992

Antibiotic resistance of the subgingival microbiota following local tetracycline therapy.

The antibiotic resistance of the subgingival microbiota was studied by 3 approaches. First, we assessed the ability of subgingival isolates taken following therapy to grow on media containing tetracycline (TC). Higher percentages of TC-resistant organisms appeared at TC fiber-treated periodontal sites and within the saliva 1 week after treatment as compared with pre-treatment levels. By 1 month, the percentage of TC-resistant organisms had returned to levels comparable to those seen before treatment. In the second approach, subgingival isolates taken following therapy were grown on media without antibiotics, and isolates were selected for Gram-stain and cell morphology determination. This study indicated that subgingival sites became colonized with gram-positive cocci in the same time period that an increase of TC-resistant isolates was observed in the first study. This may account for the transient increase in TC resistance, because many gram-positive cocci are intrinsically resistant to TC. In the third approach, the antibiotic resistance of subgingival gram-negative species was determined. The predominant cultivable microbiota of 9 sites from 3 subjects were isolated immediately before and 6 months after TC fiber treatment. Gram-negative rods were characterized and tested for sensitivity to TC (minimum inhibitory concentration [MIC] 1-128 micrograms/ml), penicillin at 80 micrograms/ml, and erythromycin at 8 micrograms/ml. None of the gram-negative rods were resistant to TC (MIC greater than or equal to 16 micrograms/ml), either before or after treatment. Before treatment 98% of the gram-negative rods were susceptible to TC at 1-2 micrograms/ml and after therapy 88% were susceptible.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Changes in root canal microbiota during the development of rat periapical lesions.

Periapical lesions are reproducibly induced in rats by pulp exposure and infection from the oral cavity. Lesions expand rapidly between day 7 and day 15-20 (active phase), with slowed expansion thereafter. In the present study we characterized the root canal microbiota present during the active phase of lesion development in this system. The mandibular first molars of Sprague-Dawley rats were exposed on day 0. The teeth were extracted after 7 days (n = 10 animals) and 15 days (n = 10), and the microbiota present in roots was isolated and characterized. The number of colonies isolated per tooth was similar on day 7 (1.53 +/- 0.64 colony-forming units x 10(3)) and day 15 (1.49 +/- 0.37 colony-forming units x 10(3)). No colonies were isolated from unexposed control teeth. Anaerobic bacteria increased significantly between day 7 (24.3 +/- 5.7%) and day 15 (47.3 +/- 7.5%), and the proportion of gram-negative organisms increased from day 7 (24.3 +/- 6.1) to day 15 (46.9 +/- 6.8). The predominant bacteria included, on day 7: Streptococcus and Bacteroides species; on day 15: species of Streptococcus, Bacteroides, Prevotella, Neisseria and Peptostreptococcus. Streptococcus oralis, Streptococcus mitis, Streptococcus rattus, Bacteroides pneumosintes and Bacteroides ureolyticus were frequently isolated at both points. Although approximately the same mean number of different species (approximately 3.5) was isolated per tooth on both day 7 and 15, the overall diversity of the isolates increased on day 15.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Bone Loss↗
PubMed2005

Design and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota.

Among human faecal bacteria, many members of the Clostridium leptum subgroup are fibrolytic and butyrate producing microorganisms thereby contributing to processes important to colonic health. Yet this phylogenetic subgroup remains poorly described to date. To improve detection and description of members of the C. leptum subgroup, the Clep 866 group probe was developed. Its association with probes targeting the Clostridium viride cluster (Cvir 1414) and Eubacterium desmolans species (Edes 635) allowed for the first time the detection of all members found in this phylogenetic group in human faecal microbiota. A species-specific probe was also designed to detect members of the Ruminococcus callidus species (Rcal 733). The design of signature regions was based on alignment of 16S rRNA sequences isolated from faeces of five healthy adults. Furthermore, an oligonucleotide competitor strategy was developed in order to improve the specificity of the probes formerly validated or designed in this study. The oligonucleotide probes were tested using a collection of target and non-target strains using FISH combined with flow cytometry. These new probes were added to a panel of 18 phylogenetic probes selected to describe faecal microbiota composition in 21 human faeces of healthy adults. Clostridium leptum subgroup represented 22% of the total faecal bacteria and codominated with members of Clostridium coccoides group. The cluster Faecalibacterium prausnitzii was the dominant component of the C. leptum subgroup and 20% of the latter subgroup remained unidentified at the species level.

Adult↗
PubMed2007

Spatial variation of active microbiota in the rice rhizosphere revealed by in situ stable isotope probing of phospholipid fatty acids.

This report is part of a serial study applying stable isotope labelling to rice microcosms to track the utilization of recently photosynthesized carbon by active microbiota in the rhizosphere. The objective of the present study was to apply phospholipid fatty acid-based stable isotope probing (PLFA-SIP) to detect the spatial variation of active microorganisms associated with rhizosphere carbon flow. In total, 49 pulses of 13CO2 were applied to rice plants in a microcosm over a period of 7 days. Rhizosphere soil was separated from bulk soil by a root bag. Soil samples were taken from rhizosphere and bulk soil, and the bulk soil samples were further partitioned both vertically (up layer and down layer) and horizontally with increasing distance to the root bag. Incorporation of 13C into PLFAs sharply decreased with distance to the roots. The labelling of 16:1omega9, 18:1omega7, 18:1omega9, 18:2omega6,9 and i14:0 PLFAs was relatively stronger in the rhizosphere while that of i15:0 and i17:0 increased in the bulk soil. The microorganisms associated with 16:1omega9 were active in both up- and down-layer soils. The microorganisms represented by i14:0, 18:1omega7 and 18:2omega6,9 exhibited a relatively higher activity in up-layer soil, whereas those represented by i15:0 and i17:0 were more active in down-layer soil. These results suggest that in the rhizosphere Gram-negative and eukaryotic microorganisms were most actively assimilating root-derived C, whereas Gram-positive microorganisms became relatively more important in the bulk soil. The active populations apparently differed between up- and down-layer soil and in particular changed with distance to the roots, demonstrating systematic changes in the activity of the soil microbiota surrounding roots.

Biodiversity↗
PubMed2006

Dendritic cells in the recognition of intestinal microbiota.

Mucosal dendritic cells (DCs) constantly survey the luminal microenvironment which contains commensal microbiota and potentially harmful organisms regulating pathogen recognition and adaptive as well as innate defense activation. Distinct mechanisms are beginning to emerge by which intestinal antigen sampling and handling is achieved ensuring specificity and contributing to redundancy in pathogen detection. Distinct DC subsets are associated with these mechanisms and regulate specific innate or adaptive immune responses to help distinguish between commensal microbiota, pathogens and self antigens. Understanding DC biology in the mucosal immune system may contribute to the unraveling of infection routes of intestinal pathogens and may aid in developing novel vaccines and therapeutic strategies for the treatment of infectious and inflammatory diseases.

Animals↗
PubMed2001

Evaluation of the denitrifying microbiota of anoxic reactors.

Removal of inorganic nitrogen compounds from wastewaters can be accomplished by a combination of the biological processes of nitrification and denitrification. The information on the microbiota present in denitrifying reactors is still scarce. In the present work the evaluation of the denitrifying microbiota of different reactor sludges was performed by specific activity measurements and MPN count of denitrifiers. We also present the isolation and physiological and phylogenetic characterisation of denitrifying bacteria from the anoxic reactor of a combined system treating landfill leachate. Specific denitrifying activity measurements were faster to perform and more reliable than MPN enumerations. 16S rDNA characterisation of the isolates showed that they belonged to the genera Thauera, Acidovorax and Alcaligenes and were closely related to microorganisms retrieved from ecosystems rich in recalcitrant compounds. Two of the isolates could grow on aromatic compounds as sole carbon source.

Journal Article↗
PubMed1985

Sampling of subgingival microbiota for dark-field microscopy.

The aim of the present investigation was to study the validity of 2 different methods of sampling the subgingival microbiota. The composition of the flora was estimated using the dark-field microscope. Subgingival samples were obtained using either a curette or a Hamilton microsyringe for a subgingival washing. The method of determination of the composition of the subgingival microorganisms was found to be very reproducible when the same investigator performed the counting. Samples obtained by the washing technique had a slight but significantly higher proportion of coccoid cells when compared to samples obtained by curette. The calculation of the reproducibility of the washing technique as assessed in 6 immediately repeated samples from the same sites yielded a coefficient of variation of 40% for spirochetes and motile rods. When 9 repeated samples from each of 28 sites (14 with a curette, 14 with washing) were studied over a period of 32 days, no systemic alterations were observed in the % of spirochetes and motile rods. The variation within each individual site was high. For curette samples and washing samples, the coefficients of variation for spirochetes and motile rods were 85% and 63%, respectively. The present investigation clearly demonstrated that care should be taken in the interpretation of single samples of subgingival microbiota. A more relevant picture of the actual situation is achieved when trends of repeated samples over time are studied.

Bacteria↗
PubMed1988

Effect of modified Widman flap surgery and systemic tetracycline on the subgingival microbiota of periodontal lesions.

33 subjects with evidence of active destructive periodontal disease were treated by modified Widman flap surgery and systemic tetracycline (1 g/day for 21 days). Subgingival plaque samples were taken from 41 sites in 12 of these subjects before and 6 months after therapy for predominant cultivable microbiota studies. Mean pocket depth and attachment levels in the 41 sampled sites were 7.1 +/- 2.9 mm and 7.7 +/- 3.2 mm prior to therapy and 4.8 +/- 2.3 mm and 6.2 +/- 3.4 mm after therapy. B. melaninogenicus and V. parvula were more frequently detected in samples taken after therapy, while S. intermedius, S. morbillorum, S. uberis and W. recta were less frequently detected after therapy. A. actinomycetemcomitans were detected in 7 sites pretherapy and 1 site post therapy. The frequency of detection of B. gingivalis and B. intermedius was virtually unchanged. The mean levels of the Actinomyces sp., A. actinomycetemcomitans, B. gingivalis, B. intermedius, S. morbillorum, S. uberis and W. recta were decreased after therapy, while the mean levels of B. melaninogenicus, S. mitis, S. sanguis II and V. parvula were increased after therapy. V. parvula showed the greatest increase to 8.2% of the microbiota. In the second phase of the study, subgingival plaque samples from 94 sites in the 33 treated subjects were analyzed by predominant cultivable techniques. As a result of therapy, 24 sites exhibited attachment loss greater than 2 mm, 23 sites exhibited "gain" greater than 2 mm and the remaining 47 sites were considered to be unchanging.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1989

The microbiota of early periodontitis lesions in adults.

The predominant cultivable microbiota of shallow progressive (active) periodontal lesions in 4 adults (29-40 years) was compared to that of shallow inactive sites. Periodontal attachment levels were monitored longitudinally, and sites showing 2-3 mm attachment loss in the previous 1-2 months were considered disease active. Inactive sites showed no change in attachment level. The microbiota of shallow sites was dominated by gram negative rod species including Wolinella recta, Bacteroides forsythus, and Fusobacterium nucleatum. W. recta was isolated in higher incidence and proportions from the active compared to inactive sites, and showed a positive relationship to bleeding on probing. Cluster analysis of species isolated from sites grouped sites from each individual together. This preliminary investigation suggested that a similar range of pathogens may be related to early periodontitis in adults as that previously reported for advanced diseased sites, and that different subjects may demonstrate different periodontal infections.

Adult↗
PubMed1990

Changes in subgingival microbiota during puberty. A 4-year longitudinal study.

It was the purpose of the present investigation to monitor the composition of the subgingival microbiota at selected sites in individuals passing through puberty and to correlate observed changes with the development of pubertal maturation. Between the ages of 11 and 14 years, pubertal and skeletal maturation was monitored annually in 22 boys and 20 girls. During this time, subgingival microbial samples were taken every 4th to 5th month (10 times in 4 years) mesially of the upper first molars. High values in total bacterial counts were reached after the onset of puberty, followed by a decrease towards the end of the observation period. The frequency of detection of Actinomyces odontolyticus and of Capnocytophaga sp. increased with time. The frequencies of other selected species, specifically of black pigmenting Bacteriodes sp. were not found to increase when tested by linear and quadratic models of time trend. However, a statistically significant rise in the frequency of detecting B. intermedius and B. melaninogenicus was noted in the initial pubertal phase identified by the onset of testicular growth in boys (p = 0.05). A significant relationship also existed between testes growth and increase of A. odontolyticus (p less than 0.01). In girls, a similar increase was obtained for A. odontolyticus when studied in relation to the Tanner scores for breast development (p less than 0.01). The changes observed in the subgingival microbiota during puberty may be related to the development of gingivitis, which was demonstrated by a higher tendency for gingival bleeding during the course of the pubertal maturation process.

Actinomyces↗
PubMed1997

Comparison of the subgingival microbiota of periodontally healthy and diseased adults in northern Cameroon.

Our study is the 1st report on subgingival microbiota in adult Cameronians. The aim was to investigate, using the checkerboard DNA-DNA hybridization technique, the prevalence of 18 oral species in subgingival plaque samples obtained from sex- and age-matched Cameronian adults with and without periodontal destruction. We also compared cultivation and the Affirm DP test with the checkerboard technique in their capability to detect some selected species among the 18. 21 adult periodontitis patients and 21 periodontally healthy subjects were examined and the results were compared statistically. Each periodontitis patient had at least 4 pockets of > or = 6 mm depth, while the healthy subjects had no sites more than 3 mm deep. Results of the checkerboard analysis showed that significantly (p < 0.05) more periodontitis patients tested positive for most of the 18 bacterial species. The Gram-positive species Actinomyces naeslundii, Streptococcus mitis and Streptococcus sanguis, known as microbiota of healthy sites, were detected significantly more frequently in the healthy group. Cultivation demonstrated P. gingivalis, B. forsythus, A. actinomycetemcomitans, P. intermedia and F. nucleatum in significantly lower %s of patients as compared to the checkerboard technique. Furthermore, the Affirm DP test detected P. gingivalis and B. forsythus in significantly fewer patients than did the checkerboard technique. A. actinomycetemcomitans was detected in 52.3% of the patients by the latter technique while the Affirm DP test failed to detect the bacterium in any of the samples. Overall, the results of the present study confirm the importance of the screening method and indicate that the prevalences of the investigated putative periodontal pathogens and beneficial species in the healthy and diseased adult Cameronians were similar to those reported for adults in the West and in some developing countries.

Actinomyces↗