Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10(7) cells g (dry weight) of feces(-1). The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.     

Effects of Alternative Dietary Substrates on Competition between Human Colonic Bacteria in an Anaerobic Fermentor System

Duplicate anaerobic fermentor systems were used to examine changes in a community of human fecal bacteria supplied with different carbohydrate energy sources. A panel of group-specific fluorescent in situ hybridization probes targeting 16S rRNA sequences revealed that the fermentors supported growth of a greater proportion of Bacteroides and a lower proportion of gram-positive anaerobes related to Faecalibacterium prausnitzii, Ruminococcus flavefaciens-Ruminococcus bromii, Eubacterium rectale-Clostridium coccoides, and Eubacterium cylindroides than the proportions in the starting fecal inoculum. Nevertheless, certain substrates, such as dahlia inulin, caused a pronounced increase in the number of bacteria related to R. flavefaciens-R. bromii and E. cylindroides. The ability of three strictly anaerobic, gram-positive bacteria to compete with the complete human fecal flora was tested in the same experiment by using selective plating to enumerate the introduced strains. The Roseburia-related strain A2-183^F was able to grow on all substrates despite the fact that it was unable to utilize complex carbohydrates in pure culture, and it was assumed that this organism survived by cross-feeding. In contrast, Roseburia intestinalis L1-82^R and Eubacterium sp. strain A2-194^R survived less well despite the fact that they were able to utilize polysaccharides in pure culture, except that A2-194^R was stimulated 100-fold by inulin. These results suggest that many low-G+C-content gram-positive obligate anaerobes may be selected against during in vitro incubation, although several groups were stimulated by inulin. Thus, considerable caution is necessary when workers attempt to predict the in vivo effects of probiotics and prebiotics from their effects in vitro.     

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 × 10¹⁰ cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 × 10¹⁰ 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 × 10⁷ to 7 × 10⁸ 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.     

Extensive Set of 16S rRNA-Based Probes for Detection of Bacteria in Human Feces

For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora. Probes were designed for Phascolarctobacterium and relatives (Phasco741), Veillonella (Veil223), Eubacterium hallii and relatives (Ehal1469), Lachnospira and relatives (Lach571), and Eubacterium cylindroides and relatives (Ecyl387), and two probes were designed for Ruminococcus and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730 and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90% of the total bacterial cells.     

Bifidobacteria in Feces and Environmental Waters

Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays showed that Bifidobacterium adolescentis, B. bifidum, B. dentium, and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve, B. infantis, and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 bifidobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured bifidobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Bifidobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host specificity. Using species-specific assays, bifidobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of bifidobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable.     

Oligonucleotide Probes That Detect Quantitatively Significant Groups of Butyrate-Producing Bacteria in Human Feces

16S rRNA-targeted oligonucleotide probes were designed for butyrate-producing bacteria from human feces. Three new cluster-specific probes detected bacteria related to Roseburia intestinalis, Faecalibacterium prausnitzii, and Eubacterium hallii at mean populations of 2.3, 3.8, and 0.6%, respectively, in samples from 10 individuals. Additional species-level probes accounted for no more than 1%, with a mean of 7.7%, of the total human fecal microbiota identified as butyrate producers in this study. Bacteria related to E. hallii and the genera Roseburia and Faecalibacterium are therefore among the most abundant known butyrate-producing bacteria in human feces.     

Diversity and abundance of the bacterial 16S rRNA gene sequences in forestomach of alpacas (Lama pacos) and sheep (Ovis aries)

Two bacterial 16S rRNA gene clone libraries were constructed from the forestomach of alpacas and sheep fed alfalfa. After the amplification using the universal 16S rRNA gene primers, equal quantities of PCR products from the same species were mixed and used to construct the two libraries. Sequence analysis showed that the 60 clones from alpacas were divided into 27 phylotypes with 25% clones affiliated with Eubacterium sp. F1. The 60 clones from sheep were divided into 21 phylotypes with 7 phylotypes affiliated with Prevotella ruminicola (40% clones). Clones closely related to Clostridium proteoclasticum, Eubacterium sp. F1, Clostridium cellobioparum, Mogibacterium neglectum, Eubacterium ventriosum, Clostridiaceae bacterium WN011, Clostridium coccoides, Clostridium orbiscindens, Eubacterium sp. F1, Cytophaga sp. Dex80-37, Treponema bryantii and Pelotomaculum sp. FP were only found in the forestomach of alpacas, and those to Anaerovorax odorimutans, Treponema zioleckii, Bifidobacterium indicum, Paludibacter propionicigenes, Paraprevotella clara, Eubacterium siraeum, Desulfotomaculum sp. CYP1, Clostridium bolteae, Clostridium termitidis and Clostridiaceae bacterium DJF_LS40 only in the rumen of sheep. Quantitative real-time PCR revealed that the forestomach of alpacas had significantly lower density of bacteria, with bacterial 16S rRNA gene copies (6.89 [Log10 (copies per gram of wet weight)]), than that of sheep (7.71, P < 0.01). The two clone libraries also appeared different in Shannon index (library from alpacas 3.30 and from sheep 3.04). Our results showed that there were apparent differences in the bacterial diversity and abundance in the forestomach between alpacas and sheep.     

Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution

A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.     

Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.     

Molecular Detection of Campylobacter spp. and Fecal Indicator Bacteria during the Northern Migration of Sandhill Cranes (Grus canadensis) at the Central Platte River

The risk to human health of the annual sandhill crane (Grus canadensis) migration through Nebraska, which is thought to be a major source of fecal pollution of the central Platte River, is unknown. To better understand potential risks, the presence of Campylobacter species and three fecal indicator bacterial groups (Enterococcus spp., Escherichia coli, and Bacteroidetes) was assayed by PCR from crane excreta and water samples collected during their stopover at the Platte River, Nebraska, in 2010. Genus-specific PCR assays and sequence analyses identified Campylobacter jejuni as the predominant Campylobacter species in sandhill crane excreta. Campylobacter spp. were detected in 48% of crane excreta, 24% of water samples, and 11% of sediment samples. The estimated densities of Enterococcus spp. were highest in excreta samples (mean, 4.6 × 10⁸ cell equivalents [CE]/g), while water samples contained higher levels of Bacteroidetes (mean, 5.1 × 10⁵ CE/100 ml). Enterococcus spp., E. coli, and Campylobacter spp. were significantly increased in river water and sediments during the crane migration period, with Enterococcus sp. densities (∼3.3 × 10⁵ CE/g) 2 to 4 orders of magnitude higher than those of Bacteroidetes (4.9 × 10³ CE/g), E. coli (2.2 × 10³ CE/g), and Campylobacter spp. (37 CE/g). Sequencing data for the 16S rRNA gene and Campylobacter species-specific PCR assays indicated that C. jejuni was the major Campylobacter species present in water, sediments, and crane excreta. Overall, migration appeared to result in a significant, but temporary, change in water quality in spring, when there may be a C. jejuni health hazard associated with water and crops visited by the migrating birds.     

Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ²⁸. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.     

Use of Rolling Circle Amplification to Rapidly Identify Species of Cladophialophora Potentially Causing Human Infection

The genus Cladophialophora comprises etiologic agents of disease in immunocompetent patients, ranging from mild cutaneous colonization to cerebral encephalitis, in addition to saprobic species. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent. In the present study, we described rapid and sensitive rolling circle amplification (RCA) method based on species-specific padlock probes targeted for the internal transcribed spacer regions of rDNA. ITS regions of 12 Cladophialophora species were sequenced, and subsequently, 10 specific padlock probes were designed for the detection of single nucleotide polymorphisms. The majority of circularizable padlock probes were designed based on single nucleotide polymorphisms (SNPs), while for C. bantiana, C. immunda and C. devriesii were characterized by two or more nucleotides. Individual species-specific probes correctly identified in all ten Cladophialophora species correctly by visualization on 1.2 % agarose gels used to verify specificity of probe-template binding; no cross-reactivity was observed. Simplicity, sensitivity, robustness and low costs provide RCA a distinct place among isothermal techniques for DNA diagnostics. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications.     

Comparison between Cultured Small-Intestinal and Fecal Microbiotas in Beagle Dogs

The microbiota of the small intestine is poorly known because of difficulties in sampling. In this study, we examined whether the organisms cultured from the jejunum and feces resemble each other. Small-intestinal fluid samples were collected from 22 beagle dogs with a permanent jejunal fistula in parallel with fecal samples. In addition, corresponding samples from seven of the dogs were collected during a 4-week period (days 4, 10, 14, and 28) to examine the stability of the microbiota. In the jejunal samples, aerobic/facultative and anaerobic bacteria were equally represented, whereas anaerobes dominated in the fecal samples. Despite lower numbers of bacteria in the jejunum (range, 10² to 10⁶ CFU/g) than in feces (range, 10⁸ to 10¹¹ CFU/g), some microbial groups were more prevalent in the small intestine: staphylococci, 64% versus 36%; nonfermentative gram-negative rods, 27% versus 9%; and yeasts, 27% versus 5%, respectively. In contrast, part of the fecal dominant microbiota (bile-resistant Bacteroides spp., Clostridium hiranonis-like organisms, and lactobacilli) was practically absent in the jejunum. Many species were seldom isolated simultaneously from both sample types, regardless of their overall prevalence. In conclusion, the small intestine contains a few bacterial species at a time with vastly fluctuating counts, opposite to the results obtained for the colon, where the major bacterial groups remain relatively constant over time. Qualitative and quantitative differences between the corresponding jejunal and fecal samples indicate the inability of fecal samples to represent the microbiotas present in the upper gut.     

Quantification of the Flavonoid-Degrading Bacterium Eubacterium ramulus in Human Fecal Samples with a Species-Specific Oligonucleotide Hybridization Probe

To investigate the occurrence of the flavonoid-degrading bacterium Eubacterium ramulus in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E.ram-0997-a-A-18 to fecal samples from 20 subjects indicated the presence of E. ramulus in each individual tested in numbers from 4.4 × 10⁷ to 2.0 × 10⁹ cells/g of fecal dry mass. Six fecal E. ramulus isolates were recognized by S-S-E.ram-0997-a-A-18 but exhibited different band patterns when analyzed by randomly amplified polymorphic DNA.     

Klebsiella Biotypes Among Coliforms Isolated from Forest Environments and Farm Produce

Samples of water, soil, needles, and bark were collected from three different forest environments and from a pulp and paper mill. In addition, samples of fresh produce were obtained from a local supermarket. All samples were examined for total and fecal coliforms. The counts obtained from the forestrelated samples did not correlate with sample type or location. When 123 isolates were identified biochemically, 71% were Klebsiella, 19% Enterobacter, 8% Citrobacter, and 2% Escherichia. All the Citrobacter, 75% of the Enterobacter, and 65% of the Klebsiella were negative for growth in elevated coliform (EC) broth. All the Escherichia were EC positive. The counts obtained from the fresh produce were generally higher than the forest counts, but the distribution of biotypes was similar. Of the 146 isolates examined 64% were Klebsiella, 14% were Escherichia, 14% were Enterobacter, and 8% were Citrobacter. All the Enterobacter and Citrobacter were EC negative, whereas 25% of the Klebesiella and 80% of the Escherichia were EC positive.     

Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r² = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

Detection of periodontopathogenic bacteria in pregnant women by traditional anaerobic culture method and by a commercial molecular genetic method

To culture facultative and strict anaerobic bacteria is a well-established method for analyzing subgingival plaque samples. Micro-IDent^® and micro-IDent^® Plus (HAIN Lifescience GmbH, Nehren, Germany) tests are two commercially available rapid PCR-based methods for the identification and quantification of putative periodontopathogen bacteria. In this study, we compared these commercial PCR-based hybridization methods with conventional anaerobic culture technique. A total of 36 subgingival plaque samples were collected from periodontal pockets of pregnant women with chronic localized periodontitis. Aliquots of these samples were evaluated with species-specific probes provided by micro-IDent^® and micro-IDent^® Plus tests simultaneously, and from the same samples anaerobic and capnophylic bacteria were cultured on selective media. The overall agreement between both methods was excellent for Eubacterium nodatum, Tannerella forsythia and Porphyromonas gingivalis (97–92%), fair for Capnocytophaga sp, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Prevotella intermedia (91–89%) and poor for Fusobacterium nucleatum, Parvimonas micra (Micromonas micros), and Campylobacter rectus (86–78%). Discrepancies in the results may be explained by inability of culture method to distinguish between closely related taxa (e.i P. intermedia/Prevotella. nigrescens), and problems of keeping periodontopathogen bacteria viable, which is required for successful detection by standard culture method. Nucleic acid-based methods may replace cultivation method as frequently used methods in microbiological diagnosis of progressive periodontitis, thus micro-IDent^® andmicro-IDent^® Plus tests can be recommended where culture of periodontopathogenic bacteria is not performed in routine microbiology laboratories to analyze subgingival plaque samples.     

Distribution of Escherichia coli O157 in Bovine Fecal Pats and Its Impact on Estimates of the Prevalence of Fecal Shedding

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g⁻¹ between samples from the same fecal pat. The density in most positive samples was <100 CFU g⁻¹, the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.