Effect of sodium monensin and cinnamaldehyde on the growth and phenotypic characteristics of Prevotella bryantii and Prevotella ruminicola.

Two representative strains of Gram-negative rumen bacteria from the genus Prevotella were used as model organisms in order to evaluate the effect of cinnamaldehyde (the secondary metabolite found in extracts of the Cinnamomum family) vs. sodium monensin on growth, cell size and cell protein production. Prevotella bryantii B(1)4 was found to be remarkably more resistant to the action of both compounds than Prevotella ruminicola 23. The approximate IC(50) concentrations of sodium monensin influenced the increase in cell size of both strains during growth, which was much more pronounced in the case of the B(1)4 strain. A similar effect was observed in strain B(1)4 when 1.438 mmol/L cinnamaldehyde was added to the growth medium, indicating a possible interference with cell division. The action of cinnamaldehyde on P. bryantii B(1)4 was concentration-dependent, in contrast to the effect observed on P. ruminicola 23.

Diet-dependent shifts in ruminal butyrate-producing bacteria.

The influence of a host's diet on Butyrivibrio and Pseudobutyrivibrio populations was investigated by competitive PCR. Specific primers were designed and competitive PCRs developed for both groups. Results (from 4 cows with different diets) suggested that high-fiber intake essentially increases the Butyrivibrio amounts in the rumen, whereas high-energy food additives lead to its suppression. The Pseudobutyrivibrio concentration also changed during the experiment but without any significant relation to the host's diet.

The effects of plant extracts on microbial community structure in a rumen-simulating continuous-culture system as revealed by molecular profiling.

An in vitro study in dual-flow continuous-culture fermentors was conducted with two different concentrations of monensin, cinnamaldehyde or garlic extract added to 1:1 forage-to-concentrate diet in order to determine their effects on selected rumen bacterial populations. Samples were subjected to total DNA extraction, restriction analysis of PCR amplified parts of 16S rRNA genes (ARDRA) and subsequent analysis of the restriction profiles by lab-on-chip technology with the Agilent's Bioanalyser 2100. Eub338-BacPre primer pair was used to select for the bacteria from the genera Bacteroides, Porphyromonas and Prevotella, especially the latter representing the dominant Gram-negative bacterial population in the rumen. Preliminary results of HaeIII restriction analysis show that the effects of monensin, cinnamaldehyde and garlic extract on the BacPre targeted ruminal bacteria are somewhat different in regard to targeted populations and to the nature of the effect. Garlic extract was found to trigger the most intensive changes in the structure of the BacPre targeted population. Comparison of the in silico restriction analysis of BacPre sequences deposited in different DNA databanks and of the results of performed amplified ribosomal DNA restriction analysis showed differences between the predicted and obtained HaeIII restriction profiles, and suggested the presence of novel, still unknown Prevotella populations in studied samples.

Obstacles to flow cytometric analysis of rumen microbial samples.

Several methods were tested that would improve the fluorescence signal from hybridized rumen bacterial cells. Disruption of cell envelopes by lysozyme, EDTA, proteinase K and/or SDS caused only a minor increase in fluorescence signal. Use of helper unlabeled oligonucleotide probes was successful only with the Puni[H672] probe which, however, when used with specific PBBl4-labeled probe, gave fluorescence signal drop. No substantial rise in fluorescence signal was also observed with cells subjected to growth-without-cell-division treatment. Further improvements are needed to make the fluorescent in situ hybridization (FISH)-flow cytometry combination applicable to rumen bacteria.

Anaerobic bacteria in the gut of terrestrial isopod Crustacean Porcellio scaber.

Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and Enterococcus were retrieved, as well as sequences from 'A1 subcluster' of the wall-less mollicutes. Bacteria from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable, i.e. predominantly oxic, conditions.

Molecular microbiology of gut bacteria: genetic diversity and community structure analysis.

Recently developed molecular biology approaches make possible the detailed genetic, taxonomic and ecological examination of microorganisms from various habitats. Animal gut represents one of the most complex microbial ecosystems with a large degree of microbial biodiversity present. Bacteria inhabiting the gut usually play important roles in metabolic transformations of substrates and sometimes, e.g. in ruminants, they make the basis for an obligate symbiosis with the host. Here we discuss molecular microbiology as a strategy for examination of gut bacteria, concentrating on a typical and in such environment dominant group of strictly anaerobic Gram-negative bacteria from the phylogenetic group Cytophaga/Flexibacter/Bacteroides. The bacteria from the genus Prevotella are the most abundant Gram-negative bacteria in the rumen and form a distinctive phylogenetic cluster, clearly separated from prevotellas isolated from other ecological niches. They may represent a good choice for a model organism in genetic manipulation experiments and for studies of gene transfer mechanisms taking place in the gut. The molecular tools for detection and monitoring of ruminal prevotellas are discussed.

Non-specific DNAases from the rumen bacterium Prevotella bryantii.

Extracellular non-specific nucleases were observed in some strains belonging to the ruminal species of the genus Prevotella, mostly P. brevis and P. bryantii. The nuclease from P. bryantii appeared to be extracellular; it mediates the degradation of the supercoiled plasmid DNA via an open circle intermediate. The cleavage is not site specific although a preference for certain cleavage sites does seem to exist. Our attempts to clone the wild-type P. bryantii B(1)4 nuclease in E. coli strain ER1992 that reports on the DNA damage sustained, were unsuccessful probably due to excessive intracellular nuclease activity that killed the cells bearing the gene for the nuclease. On the other hand, the nuclease from a related strain TCl-1, which has a less active enzyme of the same type, was successfully cloned.

The bacteriophages of ruminal prevotellas.

Rumen bacteriophage-lyzed bacterial strains of the genus Prevotella were isolated and preliminarily characterized. The strain TCl-1 the species P. bryantii was the only prevotella strain successfully infected with filter sterilized rumen fluid from a black-and-white Holstein cow. Two types of plaques were observed, both rather small and turbid. Preliminary electron microscopy observation showed that several morphologically different bacteriophages were present in these plaques. The plaque eluates were further used for the infection of other prevotella strains. The plaques produced by the bacteriophages were observed with two strains, i.e. P. bryantii B(1)4 and P. brevis GA33. The bacteriophages from both strains were examined by transmission electron microscopy and several morphologically different bacteriophages were observed, among others also a large virion with an icosahedral head with the diameter of approximately 120 nm. The bacteriophage was identified in plaques of bacterial cells of the strain GA33 and has an approximately 800 nm long helical tail, which places it among the largest ruminal bacteriophages described to date. Other bacteriophages from the same indicator strain as well as from P. bryantii B(1)4 strain were smaller and tail structures were not observed in all of them.

Systematics and evolution of ruminal species of the genus Prevotella.

Bacterial species of the genus Prevotella represent a numerically dominant microbial population in the rumen of cattle. They belong to the phylogenetic division Cytophaga-Flexibacter-Bacteroides (CFB) which is a large group of ecologically diverse bacteria with only a few shared traits. The phylogenetic descent from a common ancestor seems to be unquestionable, however, as judged from the small subunit ribosomal RNA analysis. Only 4 ruminal Prevotella species have been described to date, even though the sequence analysis of directly retrieved 16S rRNA genes indicates a large genetic diversity within this group of rumen bacteria. The closest relatives of ruminal Prevotella spp. are not surprisingly other species of the genus Prevotella, typically inhabiting the gastrointestinal tract, oral cavity and genital areas of other animals and man. The previous phylogenetic analysis showed that species of the genus Prevotella can be split into two groups or superclusters, the "ruminal" and the "non-ruminal prevotellas". One of 4 currently described ruminal Prevotella spp., i.e. P. albensis, has been placed outside the supercluster containing ruminal Prevotella spp. and within the supercluster containing the non-ruminal Prevotella spp. However, the number of available small subunit rRNA sequences from this species represents only a fraction of all known ruminal Prevotella sequences.

Application of flow cytometry for ecological monitoring of the rumen microbial ecosystem.

Flow cytometry in combination with fluorescently labeled ribosomal RNA oligonucleotide probes was used for enumeration and monitoring of ruminal bacteria. The polyanionic azo dye Trypan Blue was used for discrimination between live bacterial cells and inorganic particles and the separation was further improved by lysozyme treatment and sonication. Cy3-labeled universally conserved probe EUB338 and FITC-labeled Prevotella bryantii specific probe PBB14 were used for in situ hybridization in mixed culture experiments and in samples of crude rumen fluid. The results were analyzed by flow cytometry. The separation of P. bryantii and Butyrivibrio fibrisolvens, another ruminal bacterium, in mixed culture experiments was satisfactory and enabled monitoring of these bacteria in a test system. P. bryantii cells were detected in crude rumen fluid samples only after supplementation with pure culture cells; this implicates a low concentration of P. bryantii cells in vivo (less than 100/nL, i.e. 10(5) per mL).

Ribosomal RNA genes from Prevotella bryantii: organization and heterogeneity.

Five P. bryantii B(1)4 16S rRNA gene copies and their flanking regions were cloned and analyzed. A genomic library was constructed and screened with oligonucleotide DNA probe specific for 16S rRNA gene of P. bryantii. Five out of six different copies of 16S RNA gene were recovered and sequenced. Only minor differences (0.3-1.2%) between copies were detected within the 1541 bp long sequence. The impact of the sequence variability of 16S rRNA gene copies on phylogenetic positioning of P. bryantii was determined. All five sequences from cloned P. bryantii B(1)4 16S rRNA genes were placed in the same operational taxonomy unit. Control regions of all five analyzed rRNA operons were almost identical and three candidate for promoter sequences were identified by Neutral Network Promoter Prediction. Spacer regions between 16S rRNA and 23S rRNA genes in all five cloned copies were 543 bp long and genes for tRNA(Ile) and tRNA(Ala) were identified inside this regions.

Molecular monitoring of ruminal prevotellas.

The development and preliminary use of two different molecular approaches for rapid enumeration and monitoring of ruminal prevotellas are described. Several oligonucleotide DNA probes, specific for the genus Prevotella and the species P. ruminicola and P. bryantii were labeled with various fluorochromes and used in in situ hybridization experiments. Epifluorescent microscopy was successfully used for the detection of fluorescent signal emitted by the probes in pure and mixed culture samples. The enumeration of the target cells and the analysis of the crude rumen fluid proved to be difficult, however, mainly due to the autofluorescent background and nonhomogeneous distribution of the cells on the microscope slide. The development of a competitive PCR system for ruminal prevotellas is described and the preliminary results of the rumen fluid analysis from a black-and-white Holstein cow are given.

Preliminary characterization of a tentatively novel rumen bacterial species from the genus Treponema.

A new spirochetal strain was isolated from the rumen of a black-and-white Holstein cow and preliminarily characterized. The sugar fermentation tests and morphological observations indicated this organism to be a member of a novel, as yet undescribed spirochetal rumen species. The small subunit ribosomal RNA genes were amplified and the PCR products were cut with the restriction endonucleases TaqI, DdeI, HhaI and Sau3AI. The comparison of the observed RFLP with the hypothetical fragment lengths of the computer analyzed 16S rRNA sequences from the type strains of the ruminal spirochetes Treponema bryantii and T. saccharophilum confirmed the tentative novel identification. Transmission electron microscopy showed that the bacterium has the typical spirochetal structures, i.e. the outer sheath, the protoplasmic cylinder and the axial filament (it is not yet clear how many flagella compose the filament). An additional extracellular structure was observed which appeared as an exocytoplasmic polar flagellum, approximately 2 microns long and protruding from one tip of the cell. The average size of the cells was 0.5 x 10-15 microns and the wavelengths and the amplitudes of the primary coils were 2.9 and 1.3 microns, respectively.

Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences.

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.

Random amplified polymorphic DNA analysis and demonstration of genetic variability among bifidobacteria isolated from rats fed with raw kidney beans.

A rise in bifidobacterial numbers resembling the Escherichia coli overgrowth phenomenon was observed in the rat small intestine in a feeding experiment with kidney beans. Bifidobacterial colony counts increased from 7.6 x 10(6) to 1.7 x 10(8) cfu.g-1 of intestinal tissue in the anterior part and from less than 1 x 10(5) to 2.65 x 10(8) cfu.g-1 in posterior part of the intestine. Fifteen bifidobacterial strains were purified and further analysed. Random amplified polymorphic DNA (RAPD) assays were used to genetically differentiate bifidobacterial isolates from rat gut and compare them with type strains of 20 different species from the genus Bifidobacterium. A total of 80 arbitrary decamere primers were screened with 6 isolates, and 7 primers were chosen for the final analysis. The amplified DNA bands were scored and analysed by the unweighted pair-group method using arithmetic averages clustering. The isolates were not identical to each other nor to the screened type strains. Whereas it was possible to group 12 of the isolates into 2 separate clusters, 3 strains showed no significant relatedness to any strain. The results of the RAPD analysis indicated that there was a large degree variability among the bifidobacteria in the rat gut and demonstrated the potential applicability of such an approach in the investigation of microbial diversity in complex ecosystems.

Phenotypic diversity among ruminal isolates of Prevotella ruminicola: proposal of Prevotella brevis sp. nov., Prevotella bryantii sp. nov., and Prevotella albensis sp. nov. and redefinition of Prevotella ruminicola.

Selected phenotypic characteristics of isolates of Prevotella ruminicola (formerly Bacteroides ruminicola) were studied in order to establish whether the characteristics of genotypic strain groups established previously on the basis of 16S ribosomal DNA sequences differed systematically. Among strains formerly considered P. ruminicola subsp. brevis, strains related to strain GA33T (T = type strain) typically failed to produce carboxymethyl cellulase (CMCase) activity detectable by plate assays and failed to ferment xylose, while strains related to strain B14T produced abundant CMCase and fermented xylose. We propose that strains related to GA33T, which have DNA G + C contents between 45 and 51 mol%, should be assigned to a new species, Prevotella brevis, and that strains related to B14T, which have DNA G + C contents between 39 and 43 mol%, should be assigned to another new species, Prevotella bryantii. Most of the isolates formerly classified as P. ruminicola subsp. ruminicola strains produced CMCase and had DNA G + C contents between 45 and 51 mol%, and we propose that these organisms should be placed in the redefined species P. ruminicola. A small group of isolates that have lower G + C contents are assigned to another new species, Prevotella albensis. Most P. brevis and P. bryantii strains produced abundatn extracellular DNase activity. Proteinase activities (as determined by [14C]casein hydrolysis) varied widely between strains, and P. brevis strains exhibited the highest mean activity. All strains produced dipeptidyl peptidase activity, but the relative activities against different peptide substrates exhibited by P. bryantii, P. albensis, and P. brevis differed systematically. The phenotypic differences among the newly defined species suggest that they may occupy distinct niches within the rumen ecosystem.

Genetic diversity and phylogenetic relationships among strains of Prevotella (Bacteroides) ruminicola from the rumen.

A high degree of genetic diversity among 29 strains of Prevotella (Bacteroides) ruminicola from the rumen was revealed by comparing restriction fragment length polymorphisms in 16S rRNA genes, sodium dodecyl sulfate-polyacrylamide gel profiles of total-cell proteins, and G + C contents of chromosomal DNAs. In order to obtain information on phylogenetic relationships, the sequences of a 389-bp region of the 16S rRNA gene, including variable regions 4 and 5, were compared for 10 strains. These 10 strains formed a single group when their sequences were compared with 16S ribosomal DNA sequences from other species, including Bacteroides spp. from the human colon. On the other hand, the great genetic distances between many P. ruminicola strains, including P. ruminicola subsp. brevis B(1)4 and GA33 and P. ruminicola 23T (T = type strain), support the hypothesis that these organisms should be reclassified into new species. We identified signature oligonucleotides based on 16S ribosomal DNA sequences that distinguished strains related to strains 23T, B(1)4, GA33, and M384, as well as an oligonucleotide that specifically recognized all but one of the Bacteroides and Prevotella strains tested. On the basis of the priming activities of these signature oligonucleotides in PCR reactions and on other criteria, we concluded that 12 of the original 29 strains were related to strain 23T, 4 were related to strain B(1)4, and 4 were related to strain GA33. While there are clear grounds for subdividing the species P. ruminicola on the basis of genotypic differences, it is appropriate to delay formal reclassification until further work on the phenotypic differentiation of the new groups is completed.

Distribution of xylanase genes and enzymes among strains of Prevotella (Bacteroides) ruminicola from the rumen.

The distribution of two xylanase genes was examined by Southern hybridization among 26 strains of the rumen anaerobic bacterium Prevotella (Bacteroides) ruminicola. Hybridization with a xylanase/endoglucanase gene from the type strain 23 was found in six strains while hybridization with a xylanase gene from strain D31d was found in 14 strains. Sequences related to both genes were present, on different restriction fragments, in six strains, whereas no hybridization to either gene was detected in five other strains capable of hydrolysing xylan, or in seven strains that showed little or no xylanase activity. Zymogram analyses of seven xylanolytic strains of P. ruminicola demonstrated interstrain variation in the apparent molecular masses of the major xylanases and carboxymethylcellulases that could be renatured following SDS polyacrylamide gel electrophoresis.