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Detection of bacterial DNA within meconium is often cited as evidence supporting in utero colonization. However, many studies fail to adequately control for contamination. We aimed to define the microbial content of meconium under properly controlled conditions. DNA was extracted from 141 meconium samples and subjected to cpn60-based microbiome profiling, with controls to assess contamination throughout. Total bacterial loads of neonatal meconium, infant stool, and controls were compared by 16S rRNA quantitative PCR (qPCR). Viable bacteria within meconium were cultured, and isolate clonality was assessed by pulsed-field gel electrophoresis (PFGE). Meconium samples did not differ significantly from controls with respect to read numbers or taxonomic composition. Twenty (14%) outliers with markedly higher read numbers were collected significantly later after birth and appeared more like transitional stool than meconium. Total bacterial loads were significantly higher in stool than in meconium, which did not differ from that of sequencing controls, and correlated well with read numbers. Cultured isolates were most frequently identified as Staphylococcus epidermidis, Enterococcus faecalis, or Escherichia coli, with PFGE indicating high intraspecies diversity. Our findings highlight the importance of robust controls in studies of low microbial biomass samples and argue against meaningful bacterial colonization in utero. Given that meconium microbiome profiles could not be distinguished from sequencing controls, and that viable bacteria within meconium appeared uncommon and largely consistent with postnatal skin colonization, there does not appear to be a meconium microbiota. IMPORTANCE Much like the recent placental microbiome controversy, studies of neonatal meconium reporting bacterial communities within the fetal and neonatal gut imply that microbial colonization begins prior to birth. However, recent work has shown that placental microbiomes almost exclusively represent contamination from lab reagents and the environment. Here, we demonstrate that prior studies of neonatal meconium are impacted by the same issue, showing that the microbial content of meconium does not differ from negative controls that have never contained any biological material. Our culture findings similarly supported this notion and largely comprised bacteria normally associated with healthy skin. Overall, our work adds to the growing body of evidence against the in utero colonization hypothesis.

Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.

The fungal pathogen Sclerotinia sclerotiorum (Helotiales: Sclerotiniaceae) causes white mold, a disease that leads to substantial losses on a wide variety of hosts throughout the world. This economically important fungus affects yield and seed quality, and its control mostly relies on the use of environmentally damaging fungicides. This review aimed to present the latest discoveries on microorganisms and the biocontrol mechanisms used against white mold. A special focus is put on the identification of biocontrol desirable traits required for efficient disease control. A better understanding of the mechanisms involved and the conditions required for their action is also essential to ensure a successful implementation of biocontrol under commercial field conditions. In this review, a brief introduction on the pathogen, its disease cycle, and its main pathogenicity factors is presented, followed by a thorough description of the microorganisms that have so far demonstrated biocontrol potential against white mold and the mechanisms they use to achieve control. Antibiosis, induced systemic resistance, mycoparasitism, and hypovirulence are discussed. Finally, based on our actual knowledge, the best control strategies against S. sclerotiorum that are likely to succeed commercially are discussed, including combining biocontrol desirable traits of particular interest.

Phytoplasmas ('Candidatus Phytoplasma' species) are phytopathogenic bacteria vectored by insects and are associated with crop diseases that cause severe yield losses by affecting reproductive tissue development. Infection of northern highbush blueberry plants (Vaccinium corymbosum; Ericaceae) with phytoplasma leads to yield losses by altering plant development resulting in stunting and subsequent plant death. Samples collected from symptomatic blueberry plants in two important blueberry-producing areas in Canada, in the provinces of Québec and Nova Scotia, were analysed for the presence of DNA sequences associated with phytoplasma. Analysis of the 16S rRNA gene sequences demonstrated that the plants were infected with a strain of 'Candidatus Phytoplasma asteris', which was previously identified as blueberry stunt phytoplasma (BBS; 16SrI-E). Examination of further bacterial sequences revealed that two distinct 16S rRNA-encoding gene sequences were present in each sample in combination with a single chaperonin-60 (cpn60) sequence and a single rpoperon sequence, suggesting that this strain displays 16S rRNA-encoding gene sequence heterogeneity. Two distinct rrnoperons, rrnE and the newly described rrnAI, were identified in samples analysed from all geographic locations. We propose, based on the sequences obtained, delineating the new subgroup 16SrI-(E/AI)AI, following the nomenclature proposed for heterogeneous subgroups. To our knowledge, this is the first report of a heterogeneous phytoplasma strain affecting blueberry plants and associated with blueberry stunt disease.

Phytoplasmas (species of the genus 'CandidatusPhytoplasma') are insect-vectored phytopathogenic bacteria associated with economically and ecologically important crop diseases. Strawberry production represents an important part of agricultural activity in Mexico and elsewhere, and infection of plants with phytoplasma renders the fruit inedible by altering plant development, resulting in virescence and phyllody. In this study we examined samples taken from four strawberry plants showing symptoms associated with strawberry green petal disease and from two periwinkle plants showing virescence, sampled in different areas of Mexico. Analysis of the 16S rRNA-encoding sequences showed that the plants were infected with a phytoplasma previously identified as Mexican periwinkle virescence (MPV; 16SrXIII). Examination of bacterial sequences from these samples revealed that two distinct 16S rRNA gene sequences were present in each sample along with a single chaperonin-60 (cpn60) sequence and a single rpoB sequence, suggesting that this strain displays 16S rRNA gene sequence heterogeneity. Two distinct rrn operons, identified with subgroup 16SrXIII-A and the newly described subgroup 16SrXIII-I, were identified from the six samples analyzed, delineating the novel subgroup 16SrXIII-(A/I)I, following the nomenclature proposed for heterogeneous subgroups.

An imbalance between acidogenic and methanogenic organisms during anaerobic digestion can result in increased accumulation of volatile fatty acids, decreased reactor pH, and inhibition of methane-producing Archaea. Most commonly the result of organic input overload or poor inoculum selection, these microbiological and biochemical changes severely hamper reactor performance, and there are a few tools available to facilitate reactor recovery. A small, stable consortium capable of catabolizing acetate and producing methane was propagated in vitro and evaluated as a potential bioaugmentation tool for stimulating methanogenesis in acidified reactors. Replicate laboratory-scale batch digesters were seeded with a combination of bioethanol stillage waste and a dairy manure inoculum previously observed to result in high volatile fatty acid accumulation and reactor failure. Experimental reactors were then amended with the acetoclastic consortium, and control reactors were amended with sterile culture media. Within 7 days, bioaugmented reactors had significantly reduced acetate accumulation and the proportion of methane in the biogas increased from 0.2 ± 0 to 74.4 ± 9.9 % while control reactors showed no significant reduction in acetate accumulation or increase in methane production. Organisms from the consortium were enumerated using specific quantitative PCR assays to evaluate their growth in the experimental reactors. While the abundance of hydrogenotrophic microorganisms remained stable during the recovery period, an acetoclastic methanogen phylogenetically similar to Methanosarcina sp. increased more than 100-fold and is hypothesized to be the primary contributor to reactor recovery. Genomic sequencing of this organism revealed genes related to the production of methane from acetate, hydrogen, and methanol. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7058-3) contains supplementary material, which is available to authorized users.

Chicory (Cichorium intybus) is a perennial plant (Asteraceae) that grows wild in pasture fields in Saudi Arabia. Chicory plants displaying symptoms typically induced by phytoplasmas, such as bushy phenotype and stunt, were observed in the Mulayda region, Qassim governorate, Saudi Arabia. In this study we examined samples taken from three symptomatic chicory plants and confirmed the presence of phytoplasma DNA. Analysis of the 16S rRNA-encoding sequences showed that the plants were infected with a phytoplasma from the pigeon pea witches’-broom group (16SrIX). Sequencing of the 16S rRNA-encoding gene and the partial cpn60 sequence, computer-simulated RFLP analysis, and phylogenetic analysis of both markers revealed that the phytoplasma identified was representative of a new 16SrIX-J and cpn60 UT IX-IJ subgroup. The present study identified chicory plants as a novel host for phytoplasma strains within the pigeon pea witches’-broom phytoplasma group, and expanded the known diversity of this group.

In order to address the hypothesis that seeds from ecologically and geographically diverse plants harbor characteristic epiphytic microbiota, we characterized the bacterial and fungal microbiota associated with Triticum and Brassica seed surfaces. The total microbial complement was determined by amplification and sequencing of a fragment of chaperonin 60 (cpn60). Specific microorganisms were quantified by qPCR. Bacteria and fungi corresponding to operational taxonomic units (OTU) that were identified in the sequencing study were isolated and their interactions examined. A total of 5477 OTU were observed from seed washes. Neither total epiphytic bacterial load nor community richness/evenness was significantly different between the seed types; 578 OTU were shared among all samples at a variety of abundances. Hierarchical clustering revealed that 203 were significantly different in abundance on Triticum seeds compared with Brassica. Microorganisms isolated from seeds showed 99-100% identity between the cpn60 sequences of the isolates and the OTU sequences from this shared microbiome. Bacterial strains identified as Pantoea agglomerans had antagonistic properties toward one of the fungal isolates (Alternaria sp.), providing a possible explanation for their reciprocal abundances on both Triticum and Brassica seeds. cpn60 enabled the simultaneous profiling of bacterial and fungal microbiota and revealed a core seed-associated microbiota shared between diverse plant genera.

Naturally occurring nitrogen-fixing symbionts from root nodules of caragana (Caragana arborescens) growing in central Saskatchewan were isolated following surface sterilization of caragana root nodules and squashing and spreading of the contents on yeast extract – mannitol medium. The symbiotic nature of the strains was confirmed following inoculation onto surface-sterilized C. arborescens seed in a gnotobiotic Leonard jar system. The Rhizobium isolates from C. arborescens root nodules were intermediate in generation time (g) (mean g of 5 isolates was 6.41 h) compared with the fast growers, Rhizobium leguminosarum NRG457 (g: 4.44 h), Rhizobium tropici 899 (g: 3.19 h), and Sinorhizobium meliloti BALSAC (g: 3.45 h), but they were faster than the slow-growing Bradyrhizobium japonicum USDA 110 (g: 13.86 h) and similar to Mesorhizobium amorphae (g: 7.76 h). Nitrogen derived from fixation by measuring changes in δ15N natural abundance in plant tissue confirmed the effectiveness of the strains; approximately 80% N2 from fixation. Strain identification was carried out by determining the sequences of 3 genes: 16S rRNA-encoding genes, cpn60, and recA. This analysis determined that the symbiotic partner of Canadian C. arborescens belongs to the genus Mesorhizobium and seems more related to M. loti than to previously described caragana symbionts like M. caraganae. This is the first report of Mesorhizobium sp. nodulating C. arborescens in western Canada.

Phytoplasmas are unculturable, phytopathogenic bacteria that cause economic losses worldwide. As unculturable micro-organisms, phytoplasma taxonomy has been based on the use of the 16S rRNA-encoding gene to establish 16Sr groups and subgroups based on the restriction fragment length polymorphism (RFLP) pattern resulting from the digestion of amplicon (in vitro) or sequence (in silico) with seventeen restriction enzymes. Problems such as heterogeneity of the ribosomal operon and the inability to differentiate closely related phytoplasma strains has motivated the search for additional markers capable of providing finer differentiation of phytoplasma strains. In this study we developed and validated a scheme to classify phytoplasmas based on the use of cpn60 universal target (cpn60 UT) sequences. Ninety-six cpn60 UT sequences from strains belonging to 19 16Sr subgroups were subjected to in silico RFLP using pDRAW32 software, resulting in 25 distinctive RFLP profiles. Based on these results we delineated cpn60 UT groups and subgroups, and established a threshold similarity coefficient for groups and subgroups classifying all the strains analysed in this study. The nucleotide identity among the reference strains, the correspondence between in vitro and in silico RFLP, and the phylogenetic relationships of phytoplasma strains based on cpn60 UT sequences are also discussed.

Arthrobacter sp. strain OY3WO11 inhibits the growth of the potato pathogen Phytophthora infestans in in vivo growth challenge assays. We determined the draft genome sequence of this strain, assembling it into 3 scaffolds of 4.2 Mbp total length. OY3WO11 may represent a novel species of Arthrobacter .

Pseudomonas chlororaphis strain 189 is a potent inhibitor of the growth of the potato pathogen Phytophthora infestans . We determined the complete, finished sequence of the 6.8-Mbp genome of this strain, consisting of a single contiguous molecule. Strain 189 is closely related to previously sequenced strains of P. chlororaphis .

Pantoea agglomerans isolates 3 and 4 were retrieved from the bacterial community associated with wheat seeds. These isolates differ in their pattern of growth antagonism toward Alternaria species. A comparison of the genome sequences of these two isolates revealed a high sequence identity with previously sequenced strains of P. agglomerans .

In this study, 12 strains of T hermoanaerobacter 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 T hermoanaerobacter pseudethanolicus or T hermoanaerobacter thermohydrosulfuricus . Examination of the lesser conserved chaperonin-60 ( cpn 60) universal target showed that some isolates shared the highest sequence identity with T . thermohydrosulfuricus ; however, others to T hermoanaerobacter wiegelii and T hermoanaerobacter sp. Rt8.G4 (formerly T hermoanaerobacter 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 T hermoanaerobacter isolates from a common environment.

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.

Phytoplasmas (‘Candidatus Phytoplasma’) are insect-transmitted, cell-wall-less, plant-pathogenic bacteria that cause economically important crop diseases. Because phytoplasmas are difficult or impossible to culture in vitro, they are classified taxonomically according to the convention used for unculturable micro-organisms. The first coherent scheme of classification of phytoplasmas, based on the RFLP pattern of the 16S rRNA-encoding gene generated with 17 restriction endonucleases, was updated several times until the development of the iPhyClassifier. iPhyClassifier is an interactive online tool capable of determining the species, group and subgroup of ‘Candidatus Phytoplasma’ of unknown samples using the 16S F2nR2 sequence. Latin America, an important geographical area in relation to food production, has a high incidence of plant diseases caused by phytoplasmas. However, many phytoplasmas associated with these diseases have not been properly classified. An extensive literature review and the use of iPhyClassifier allowed us to identify two new tentative groups (16SrXXXIII-A and 16SrXXXIV-A) and the following tentative new subgroups among Latin American strains that were either previously unclassified or misclassified: six in 16SrI, six in 16SrII, one in 16SrIII, one in 16SrVII, one in 16SrIX, one in 16SrXII and two in 16SrXIII.

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) (~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 (>1012genomes/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.Key words: activated sludge, biological treatment, bulking; chaperonin 60, cpn60, filamentous bacteria, mixed liquor suspended solids, microbial communities, 021N, qPCR, settling, Thiothrix.

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.

Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates exclusively in the cytoplasm of infected cells. The viral envelope glycoproteins, E1 and E2, appear to be retained in the endoplasmic reticulum, where viral budding is thought to occur. Surprisingly, we found that the expression system used to generate HCV envelope glycoproteins influences their subcellular localization and processing. These findings have important implications for optimizing novel HCV fusion and entry assays as well as for budding and virus particle formation.

Viral-bacterial co-infections are associated with severe exacerbations of COPD. Epithelial antimicrobial peptides, including human β-defensin-2 (HBD-2), are integral to innate host defenses. In this study, we examined how co-infection of airway epithelial cells with rhinovirus and Pseudomonas aeruginosa modulates HBD-2 expression, and whether these responses are attenuated by cigarette smoke and in epithelial cells obtained by bronchial brushings from smokers with normal lung function or from COPD patients. When human airway epithelial cells from normal lungs were infected with rhinovirus, Pseudomonas aeruginosa, or the combination, co-infection with rhinovirus and bacteria resulted in synergistic induction of HBD-2 (p

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.

Cellobiose dehydrogenase (CDH)-deficient strains of the basidiomycete Trametes versicolor were produced by transforming protoplasts of strain 52J with a plasmid carrying the T. versicolor cdh gene disrupted with a phleomycin resistance cassette. Of 143 phleomycin-resistant colonies analyzed, 3 did not produce measurable CDH during two successive two-week culture periods. Two of these mutants were shown to lack functional CDH when grown in CDH induction medium. They biobleached and delignified industrial unbleached kraft pulp as efficiently as did wild-type T. versicolor, indicating that CDH is not required for the degradation and biobleaching of kraft lignin. The ability to degrade 14C-guaiacyl dehydrogenative polymerizate (synthetic lignin) also appeared to be unaffected. However, compared to the parent strain, all three mutants grew poorly on minimal agar with highly crystalline cellulose as the sole carbon source. This difference was not observed on non-crystalline carbohydrates. All three mutants had a greatly decreased ability to colonize and degrade both seasoned and fresh native white birch wood, a natural substrate of T. versicolor. The dramatic decrease in T. versicolor 52J’s ability to invade and grow on birch wood caused by the loss of its secreted CDH strongly suggests that this enzyme is essential to its wood invading and degrading niche in the forest ecosystem.

Phytoplasmas (‘Candidatus Phytoplasma’ spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of ‘Ca.Phytoplasma’ spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from ‘Ca.Phytoplasma’ spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.

Protoplasts of the monokaryotic strain 52J of Trametes versicolor were treated with UV light and screened for the inability to produce a colored precipitate on guaiacol-containing agar plates. Mutants unable to oxidize guaiacol had absent or very low secretion of laccase and manganese peroxidase (MnP) proteins. All isolates unable to secrete MnP were also unable to bleach or delignify kraft pulp. One mutant strain, M49, which grew normally but did not oxidize guaiacol, was tested further with a number of other substrates whose degradation has been associated with delignification by white rot fungi. Compared with the parent, 52J, mutant M49, secreting no MnP and low laccase, could not brighten or delignify kraft pulp, produced less ethylene from 2-keto methiolbutyric acid, released much less (sup14)CO(inf2) from [(sup14)C]DHP (a synthetic lignin-like polymerizate), and produced much less methanol from pulp. This mutant also displayed decreased abilities to oxidize the dyes poly B-411, poly R-478, and phenol red compared with the wild-type strain and was also unable to decolorize kraft bleachery effluent or mineralize its organochlorine. Addition of purified MnP in conjunction with H(inf2)O(inf2), MnSO(inf4), and an Mn(III) chelator to M49 cultures partially restored methanol production, pulp delignification, and biobleaching in some cases.

Barcoding with molecular sequences is widely used to catalogue eukaryotic biodiversity. Studies investigating the community dynamics of microbes have relied heavily on gene-centric metagenomic profiling using two genes (16S rRNA and cpn60) to identify and track Bacteria. While there have been criteria formalized for barcoding of eukaryotes, these criteria have not been used to evaluate gene targets for other domains of life. Using the framework of the International Barcode of Life we evaluated DNA barcodes for Bacteria. Candidates from the 16S rRNA gene and the protein coding cpn60 gene were evaluated. Within complete bacterial genomes in the public domain representing 983 species from 21 phyla, the largest difference between median pairwise inter- and intra-specific distances (“barcode gap”) was found from cpn60. Distribution of sequence diversity along the ∼555 bp cpn60 target region was remarkably uniform. The barcode gap of the cpn60 universal target facilitated the faithful de novo assembly of full-length operational taxonomic units from pyrosequencing data from a synthetic microbial community. Analysis supported the recognition of both 16S rRNA and cpn60 as DNA barcodes for Bacteria. The cpn60 universal target was found to have a much larger barcode gap than 16S rRNA suggesting cpn60 as a preferred barcode for Bacteria. A large barcode gap for cpn60 provided a robust target for species-level characterization of data. The assembly of consensus sequences for barcodes was shown to be a reliable method for the identification and tracking of novel microbes in metagenomic studies.

The common but poorly understood condition known as bacterial vaginosis (BV) increases vulnerability to HIV infection and is associated with the absence of H(2)O(2)-producing Lactobacillus. Vaginal lactic acid bacteria (LAB) produce anti-HIV factors such as organic acids and hydrogen peroxide (H(2)O(2)), and may bind and inactivate HIV particles during scavenging of mannose. These factors define potential criteria for initial selection of candidate probiotics to block heterosexual transmission of HIV. Therefore, the primary goal of this study was to characterize acid production on mannose and H(2)O(2) production in vaginal isolates from Canadian adolescents (192 isolates, 16 individuals) and commercial sex workers in Nairobi, Kenya (576 isolates, 96 individuals). Selection of isolates from H(2)O(2)-detecting media suggested an idiosyncratic individual-level profile and extensive phenotypic diversity, including the identification of a subset of "double-strong" acid- and H(2)O(2)-producers with phenotypes similar to well-characterized probiotic strains. Molecular fingerprinting of all isolates by capillary electrophoresis of 16S-23S rRNA interspacer amplicons was coupled with chaperonin-60 universal target (cpn60 UT) sequencing in a subset, tentatively identifying 96% of isolates although only 19% were sequenced. Most isolates belonged to Lactobacillus, Streptococcus, Bifidobacterium or Gardnerella, with a total of 37 species in 15 genera, as well as 5 potentially novel organisms, identified in this study. This sensitivity was likely enhanced by phenotype-based selection on two chromogenic media formulations. Identification of double-strong isolates may provide a rational basis for selection and further characterization of vaginal probiotics, with potential application as part of HIV prevention initiatives in western Canada and East Africa.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus -dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others. This condition is associated with a range of negative health outcomes, including HIV acquisition, and it can be difficult to manage clinically. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered norma, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants. © 2011 Creative Commons Attribution License.

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.

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.

Mycobacterium avium subsp. paratuberculosis was cultured from a single fecal sample collected from a 10-yr-old, captive-bred male addax (Addax nasomaculatus). Attempts to confirm infection with additional fecal cultures, serology, semen culture, and tissue biopsy were unsuccessful. There were no gross lesions on necropsy. On histopathology there were neither acid-fast organisms nor microscopic changes suggestive of active or clinical Johne's disease. Mycobacterium avium subsp. paratuberculosis was isolated from four organ tissues: ileum, jejunum, colon, and mesenteric lymph node.

We evaluated the ability of hen-egg antibodies (HEA) to reduce intestinal colonization by Clostridium perfringens in broiler chickens. Antibodies against C. perfringens or cholera toxin (negative control) were obtained from the eggs of laying hens hyperimmunized using a C. perfringens bacterin or cholera toxin. Eggs were collected, pooled, and egg antibodies were concentrated by polyethylene-glycol precipitation. An initial experiment was conducted to determine the in vivo activity of the administered antibody along the length of the intestine. Thereafter, two feeding trials were performed to assess the efficacy of feed amended with the egg antibodies in reducing the level of colonization of C. perfringens in challenged birds. Antibody activity declined from proximal to distal regions of the intestine but remained detectable in the cecum. In the first experiment there was no significant reduction in the number of C. perfringens in the birds fed the diet amended with the anti-C. perfringens egg antibody, compared to the birds that received the anti-cholera toxin egg antibody (n = 10), at any of the sampling times. In the second experiment there was a significant decrease in C. perfringens intestinal populations 72 h after treatment (n = 15) as assessed by culture-based enumeration, but there was no decrease as measured by quantitative PCR based on the C. perfringens phospholipase C gene. Intestinal-lesion scores were higher in the birds that received the anti-C. perfringens HEA. Our work suggests that administration of HEA did not reduce the level of C. perfringens intestinal colonization and conversely might exacerbate necrotic enteritis.

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.

Seven mutations in the C2, V3, and C3 regions of gp120 are implicated in the tropism of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. Site-directed mutagenesis revealed that three amino acids are essential to maintain this tropism, one in the C2 region and two in the V3 loop. Two mutations implied N glycosylation modifications.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.