Comparison of Ileum Microflora of Pigs Fed Corn-, Wheat-, or Barley-based Diets by Chaperonin-60 Sequencing and Quantitative PCR

Applied and Environmental Microbiology. Feb, 2005  |  Pubmed ID: 15691942

We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.

Is There an Answer?

IUBMB Life. Jan, 2005  |  Pubmed ID: 16036562

Enumeration of Specific Bacterial Populations in Complex Intestinal Communities Using Quantitative PCR Based on the Chaperonin-60 Target

Journal of Microbiological Methods. Jan, 2006  |  Pubmed ID: 16112762

We used qPCR and the target gene chaperonin-60 (cpn60) to enumerate Clostridium perfringens genomes in DNA extracts from contents of the chicken gastrointestinal tract with the aim of optimizing this methodology to enumerate any bacterium of interest. To determine the most accurate protocols for determining target species abundance, we compared various DNA extraction methods in combination with four methods for producing standard curves. Factors affecting accuracy included the co-purification of PCR inhibitors and/or fluorescence quenchers and the yield of target DNA in the extract. Anion exchange chromatography of the spiked test samples enabled accurate enumeration of C. perfringens using a standard curve comprised of a plasmid containing a fragment of C. perfringens cpn60. We used qPCR to enumerate C. perfringens and other intestinal bacteria in ileum and cecum samples from chickens that had been challenged with C. perfringens and compared the results with viable counts on corresponding selective agars. We conclude that qPCR-based molecular enumeration of target species in the gastrointestinal tract is feasible, but care must be taken in order to mitigate the effects of confounding factors that can affect the apparent cell count.

Characterization of Intestinal Microbiota and Response to Dietary Virginiamycin Supplementation in the Broiler Chicken

Applied and Environmental Microbiology. Apr, 2006  |  Pubmed ID: 16597987

The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed. A total of 370 distinct cpn60 sequences were identified, which ranged in frequency of recovery from 1 to 2,872. The small intestinal libraries were dominated by sequences from the Lactobacillales (90% of sequences), while the cecum libraries were more diverse and included members of the Clostridiales (68%), Lactobacillales (25%), and Bacteroidetes (6%). To assess the effects of virginiamycin on the gastrointestinal microbiota, 15 bacterial targets were enumerated using quantitative, real-time PCR. Virginiamycin was associated with increased abundance of many of the targets in the proximal gastrointestinal tract (duodenal loop to proximal ileum), with fewer targets affected in the distal regions (ileocecal junction and cecum). These findings provide improved profiling of the composition of the chicken intestinal microbiota and indicate that microbial responses to virginiamycin are most significant in the proximal small intestine.

Molecular Characterization of Microbial Communities in Canadian Pulp and Paper Activated Sludge and Quantification of a Novel Thiothrix Eikelboomii-like Bulking Filament

Canadian Journal of Microbiology. May, 2006  |  Pubmed ID: 16699576

We examined the microbial community structure and quantified the levels of the filamentous bulking organism Thiothrix eikelboomii in samples of activated sludge mixed liquor suspended solids (MLSS) from Canadian pulp and paper mills. Libraries of chaperonin 60 (cpn60) gene sequences were prepared from MLSS total microbial community DNA and each was compared with cpnDB, a reference database of cpn60 sequences (http://cpndb.cbr.nrc.ca) for assignment of taxonomic identities. Sequences similar to but distinct from the type strain of T. eikelboomii AP3 (ATCC 49788T) (approximately 89% identity over 555 bp) were recovered at high frequency from a mill sample that was experiencing bulking problems at the time of sample collection, which corresponded to microscopic observations using fluorescent in situ hybridization with commercially available 16S rDNA-based probes. We enumerated this strain in five mill-derived MLSS samples using real-time quantitative PCR (qPCR) and found that two samples had high levels of the bulking strain (>1012 genomes/g MLSS) and two contained lower but detectable levels of this organism. None of the mill samples contained cpn60 sequences that were identical to the type strain of T. eikelboomii. This technique shows promise for monitoring pulp and paper mill wastewater treatment systems by detecting and enumerating this strain of T. eikelboomii, which may be specific to pulp and paper mill wastewater treatment systems.

Pyrosequencing of the Chaperonin-60 Universal Target As a Tool for Determining Microbial Community Composition

Applied and Environmental Microbiology. May, 2009  |  Pubmed ID: 19270139

We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.

Multiplex Detection of Bacteria Associated with Normal Microbiota and with Bacterial Vaginosis in Vaginal Swabs by Use of Oligonucleotide-coupled Fluorescent Microspheres

Journal of Clinical Microbiology. Dec, 2009  |  Pubmed ID: 19794034

Bacterial vaginosis (BV) is a recurrent condition that is associated with a range of negative outcomes, including the acquisition of human immunodeficiency virus and other sexually transmitted diseases, preterm births, and pelvic inflammatory disease. In contrast to the Lactobacillus-dominated normal vaginal microbiota, BV is characterized by a lack of lactobacilli and an abundance of anaerobic and gram-negative organisms, including Gardnerella vaginalis and Atopobium vaginae. To date, the laboratory diagnosis of BV has relied upon the fulfillment of criteria determined by microscopic observation of Gram-stained vaginal swabs. We describe a molecular-based method for the easy determination of the species profile within the vaginal microbiota based on the amplification of the chaperonin-60 genes of all bacteria present in the swab and hybridization of the amplicon to species-specific oligonucleotide-coupled fluorescent beads that are identified by flow cytometry with a Luminex instrument. We designed a nineplex Luminex array for characterization of the vaginal microbiota and applied it to the analysis of vaginal swabs from individuals from Africa and North America. Using the presence of A. vaginae or G. vaginalis, or both, as the defining criterion for BV, we found that the method was highly specific and sensitive for the diagnosis of BV using microscopy as a gold standard.

Predicting Relatedness of Bacterial Genomes Using the Chaperonin-60 Universal Target (cpn60 UT): Application to Thermoanaerobacter Species

Systematic and Applied Microbiology. May, 2011  |  Pubmed ID: 21392917

D.R. Zeigler determined that the sequence identity of bacterial genomes can be predicted accurately using the sequence identities of a corresponding set of genes that meet certain criteria [32]. This three-gene model for comparing bacterial genome pairs requires the determination of the sequence identities for recN, thdF, and rpoA. This involves the generation of approximately 4.2kb of genomic DNA sequence from each organism to be compared, and also normally requires that oligonucleotide primers be designed for amplification and sequencing based on the sequences of closely related organisms. However, we have developed an analogous mathematical model for predicting the sequence identity of whole genomes based on the sequence identity of the 542-567 base pair chaperonin-60 universal target (cpn60 UT). The cpn60 UT is accessible in nearly all bacterial genomes with a single set of universal primers, and its length is such that it can be completely sequenced in one pair of overlapping sequencing reads via di-deoxy sequencing. These mathematical models were applied to a set of Thermoanaerobacter isolates from a wood chip compost pile and it was shown that both the one-gene cpn60 UT-based model and the three-gene model based on recN, rpoA, and thdF predicted that these isolates could be classified as Thermoanaerobacter thermohydrosulfuricus. Furthermore, it was found that the genomic prediction model using cpn60 UT gave similar results to whole-genome sequence alignments over a broad range of taxa, suggesting that this method may have general utility for screening isolates and predicting their taxonomic affiliations.

Pyrosequencing of Chaperonin-60 (cpn60) Amplicons As a Means of Determining Microbial Community Composition

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21431768

The chaperonin-60 universal target (cpn60 UT) is generated from a set of PCR primers and provides a universally conserved, phylogenetically informative sequence signature for determining the composition of microbial communities by DNA sequencing. Pyrosequencing of cpn60 UT amplicons is emerging as a next-generation tool for providing unprecedented sequencing depth and resolution of microbial communities in individual samples. Owing to the increase in sequencing depth, the dynamic range across which the presence and abundance of individual species can be sampled experimentally also increases, significantly improving our ability to investigate microbial community richness and diversity. The flexible format of the pyrosequencing reaction setup combined with the ability to pool samples through the use of multiplexing IDs makes the generation of microbial profiles based on the cpn60 UT both feasible and cost-effective. We describe here the methods we have developed for determining microbial community profiles by pyrosequencing of cpn60 UT amplicons, from generating amplicons to sequencing and data analysis.

Molecular Definition of Vaginal Microbiota in East African Commercial Sex Workers

Applied and Environmental Microbiology. Jun, 2011  |  Pubmed ID: 21531840

Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV(+)) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV(+) individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV(+) women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV(+) and BV(-) diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV(+) and BV(-). Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV(-) ("normal") phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.

Biological Pretreatment with a Cellobiose Dehydrogenase-deficient Strain of Trametes Versicolor Enhances the Biofuel Potential of Canola Straw

Bioresource Technology. Nov, 2011  |  Pubmed ID: 21903381

The use of Trametes versicolor as a biological pretreatment for canola straw was explored in the context of biofuel production. Specifically, the effects on the straw of a wild-type strain (52J) and a cellobiose dehydrogenase (CDH)-deficient strain (m4D) were investigated. The xylose and glucose contents of the straw treated with 52J were significantly reduced, while only the xylose content was reduced with m4D treatment. Lignin extractability was greatly improved with fungal treatments compared to untreated straw. Saccharification of the residue of the m4D-treated straw led to a significant increase in proportional glucose yield, which was partially attributed to the lack of cellulose catabolism by m4D. Overall, the results of this study indicate that CDH facilitates cellulose access by T. versicolor. Furthermore, treatment of lignocellulosic material with m4D offers improvements in lignin extractability and saccharification efficacy compared to untreated biomass without loss of substrate due to fungal catabolism.

Isolates of Thermoanaerobacter Thermohydrosulfuricus from Decaying Wood Compost Display Genetic and Phenotypic Microdiversity

FEMS Microbiology Ecology. Dec, 2011  |  Pubmed ID: 22066958

In this study, 12 strains of Thermoanaerobacter were isolated from a single decaying wood compost sample and subjected to genetic and phenotypic profiling. The 16S rRNA encoding gene sequences suggested that the isolates were most similar to strains of either Thermoanaerobacter pseudethanolicus or Thermoanaerobacter thermohydrosulfuricus. Examination of the lesser conserved chaperonin-60 (cpn60) universal target showed that some isolates shared the highest sequence identity with T. thermohydrosulfuricus; however, others to Thermoanaerobacter wiegelii and Thermoanaerobacter sp. Rt8.G4 (formerly Thermoanaerobacter brockii Rt8.G4). BOX-PCR fingerprinting profiles identified differences in the banding patterns not only between the isolates and the reference strains, but also among the isolates themselves. To evaluate the extent these genetic differences were manifested phenotypically, the utilization patterns of 30 carbon substrates were examined and the niche overlap indices (NOI) calculated. Despite showing a high NOI (> 0.9), significant differences existed in the substrate utilization capabilities of the isolates suggesting that either a high degree of niche specialization or mechanisms allowing for non-competitive co-existence, were present within this ecological context. Growth studies showed that the isolates were physiologically distinct in both growth rate and the fermentation product ratios. Our data indicate that phenotypic diversity exists within genetically microdiverse Thermoanaerobacter isolates from a common environment.