Dr. Tim Dumonceaux’s poster
Microbiome analysis and taxonomic identification of microorganisms using the chaperonin-60 universal target.
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References
In the present study 28 Trueperella pyogenes strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene cpn60 encoding chaperonin. No cross reaction could be observed with control strains representing four species of genus Trueperella and seven species of closely related genus Arcanobacterium. The present cpn60 LAMP assay might allow a reliable and low cost identification of T. pyogenes also in laboratories with less specified equipment.
Evidence is emerging that the two chaperonin (Cpn) 60 proteins of Mycobacterium tuberculosis, Cpn60.1 and Cpn60.2, have moonlighting actions that may contribute to the pathology of tuberculosis. We studied the release of Cpn60.1 from M. tuberculosis and infected macrophagelike cells and compared recombinant Cpn60.1 and Cpn60.2 in a range of cell-based assays to determine how similar the actions of these highly homologous proteins are. We now establish that Cpns are similar as follows: (i) Cpn60.1, as it has been shown for Cpn60.2, is released by M. tuberculosis in culture, and Cpn60.1 is furthermore released when the bacterium is in quiescent, but not activated, macrophagelike cells, and (ii) both proteins only showed a partial requirement for MyD88 for the induction of proinflammatory cytokine production compared to lipopolysaccharide. However, we also found major differences in the cellular action of Cpns. (i) Cpn60.2 proved to be a more potent stimulator of whole blood leukocytes than Cpn60.1 and was the only one to induce tumor necrosis factor alpha synthesis. (ii) Cpn60.1 bound to ca. 90% of circulating monocytes compared to Cpn60.2, which bound <50% of these cells. Both chaperonins bound to different cell surface receptors, while monocyte activation by both proteins was completely abrogated in TLR4-/- mice, although Cpn60.2 also showed significant requirement for TLR2. Finally, an isogenic mutant lacking cpn60.1, but containing intact cpn60.2, was severely inhibited in generating multinucleate giant cells in an in vitro human granuloma assay. These results clearly show that, despite significant sequence homology, M. tuberculosis Cpn60 proteins interact in distinct ways with human or murine macrophages. Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Campylobacter species are important organisms in both human and animal health. The identification of Campylobacter currently requires the growth of organisms from complex samples and biochemical identification. In many cases, the condition of the sample being tested and/or the fastidious nature of many Campylobacter species has limited the detection of campylobacters in a laboratory setting. To address this, we have designed a set of real-time quantitative PCR (qPCR) assays to detect and quantify 14 Campylobacter species, C. coli, C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hyointestinalis, C. jejuni, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, and C. upsaliensis, directly from DNA extracted from feces. By use of a region of the cpn60 (also known as hsp60 or groEL) gene, which encodes the universally conserved 60-kDa chaperonin, species-specific assays were designed and validated. These assays were then employed to determine the prevalence of Campylobacter species in fecal samples from dogs. Fecal samples were found to contain detectable and quantifiable levels of C. fetus, C. gracilis, C. helveticus, C. jejuni, C. showae, and C. upsaliensis, with the majority of samples containing multiple Campylobacter species. This study represents the first report of C. fetus, C. gracilis, C. mucosalis, and C. showae detection in dogs and implicates dogs as a reservoir for these species. The qPCR assays described offer investigators a new tool to study many Campylobacter species in a culture-independent manner.
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.
Clostridium perfringens an ubiquitous environmental bacterium causes major food borne illnesses, digestive diseases and several soft tissue infections in humans and animals. In the present study, toxin typing of 91 C. perfringens isolates from animals with enteric diseases and their environments revealed the presence of type A and C strains. Enterotoxin gene (cpe), responsible for majority of the food poisoning incidences in humans and enteric infections in animals was present in 60.43 % of the isolates of which 76.3% and 23. 36% were chromosomal and plasmid borne respectively. Neighbour-joining tree inferred from cpn60 UT nucleotide sequences could differentiate the cpe+ve isolates from the cpe−ve isolates, provide clear distinction between the cpe-IS1470 and cpe-IS1151 genotypes and segregate type A and C strains in separate clusters. The present study is the first report on the utilization of cpn60 UT region for C. perfringens phylogeny analysis and demonstrates that cpn60 UT analysis alone, to a greater extent can be a simple, rapid and efficient method for differentiating between cpe+ve and cpe−ve strains or toxin types. The cpb2 gene was observed among 30 isolates of which 16.6% were from porcine sources while the rest were of non-porcine and environmental origin. The cpb2 sequences obtained in the present study though similar among them were diverse both from the consensus and atypical cpb2 sequences reported globally and formed a separate cluster. The study thus reports of novel cpb2 gene variant and warrants its characterization through further studies.
Replacement of fish meal in aquaculture diets with plant proteins is economically desirable, but the effects of alternative protein sources on the intestinal microbiota and fish health are poorly understood. We examined the intestinal microbiome of 108 rainbow trout (Oncorhynchus mykiss) fed with diets including plant ingredients (peas, soybean, canola) at two processing levels (meal, concentrate), or a fish meal (FM) control diet. Microbial community profiles were determined using pyrosequencing of . cpn60 PCR products and 16S rRNA DGGE. Microbial profiles of fish fed the FM diet changed over the course of the study with reduction in species richness and diversity and therefore evaluation of the effects of experimental diets was accomplished through comparison of each diet with its concurrent FM control. Plant ingredient diets were associated with higher Firmicutes:Proteobacteria ratios than controls. Both DGGE and pyrosequencing data showed that microbiomes of fish fed meal-based diets were more distinguishable from the FM control microbiomes than were the microbiomes of fish fed protein concentrate diets. Changes in the FM diet associated profiles could be the result of age-related physiological changes in these fish or a long-term effect of the change in environment from outdoor aquaculture facility to an indoor re-circulating facility. Regardless of the reason, these observations have significant implications for future evaluations of diets and ingredients. We also demonstrated changes in the intestinal microbiome that may contribute to negative health outcomes when diets contain plant meal proteins. These changes in microbiome structure can be minimized with additional processing of plant ingredients. © 2012 Elsevier B.V.
Douvile, M., et al. (2010). "Characterization of commercial microbial products by polymorphic DNA markers and enzymatic activity diversity: Occurrence and potential effects on freshwater mussels exposed to municipal effluents." Research Journal of Biotechnology 5(3): 31-48.
The increasing commercial applications of microbial products and consortia have raised concerns about their release to the environment and the potential for toxic effects on aquatic biota. This study characterized 25 bacteria, one yeast and one fungus using a combination of random amplified polymorphic DNA and 19 hydwlytic enzyme activities, with the purpose of determining their occurrence and potential effects on the immune systems of mussels exposed to a primary.-treated municipal effluent for seven weeks. The amplification product (552-bp) of cpn60 gene by using universal primers was sequenced for each micro-organism and available in GenBank. The microbial community structures in the digestive gland of mussels were also characterized using 16s ribosomal gene analysis by denaturing gradient gel electrophoresis (DGGE). The results revealed that each micro-organism was easily cultivated with usual growth media and the enzyme activity profiles permitted to propose an identification method using hierarchical tree and backpropagation neural networks classifications. The bacterial community structure based on DGGE of 16S rDNA in mussels changed with the concentration of the municipal effluent. This was also revealed by a characteristic change in enzyme activity profiles with increased frequency in acid and amide phosphatase, leucine aminopeptidase and ß-galactosidase in the isolated heterotrophic colonies in mussels. The analysis of heterotrophic bacteria colonies in unexposed mussels revealed that most colonies (6/10) were of unkown origin and only three bacterial colonies shared close similarities with B. subtilisis 11685, E. aerogenes, P. putida 12633. In mussels exposed to the municipal effluents, about half of the colonies were classified as unknowns while the other half was composed of bacteria showing close similarites with E. aerogenes, P. putida 12633 and 31800. These bacteria differed from those found in forming biofilms which was mainly associated to B. subtil is J J 685 or P. fluorescens 13525 functional properties. The immune system of the mussels was also compromised with decreased hemocyte density, increased phagocytosis activity and cytotoxic activity. While no trend between phagocytosis activity and the enzyme activity profiles was found, the increased frequency in leucine aminopeptidase and acid phosphatase were negatively correlated with the hemocyte density and cytotoxic activity. In conclusion, a methodology to track for the occurrence, bioavailability and potential immunotoxic effects of commercial microorganisms is proposed based on a combination of genetic and phenotypic (enzyme activity) markers and immunocompetent assessment. Preliminary results indicate mussels contained only three bacteria out of 25 bacteria that bear high functional similarity and the characteristic pertubations with the enzyme activity profiles were associated with immunocompetence changes.
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
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
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 49788 T) (~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.
This protocol describes the laboratory steps required to capture cpn60 gene fragments from microbial community DNA obtained from an environment of interest. This approach facilitates the determination of the taxonomic composition of a sample, providing information that spans all Domains of life (Bacteria, Eukarya, and Archaea). This method is independent of amplification biases associated with PCR, and provides a microbial community profile that is consistent with that generated using whole community shotgun sequencing, with substantially less sequencing effort required for the same community coverage. The protocol involves shearing the whole community genomic DNA by sonication, adding Illumina index primers, then hybridizing the sheared DNA to biotinylated RNA probes that correspond to the entire cpn60 reference database. Hybrids are selected using magnetic streptavidin beads, washed, and sequenced using an Illumina MiSeq platform. The protocol involves approximately five hours of hands-on time per sample over two days, along with a 24 hour hybridization step. In this protocol we describe the major changes we have implemented using the manufacturer’s recommendations as a starting point. We also provide tips and expected results.
The taxonomic diversity and antibiotic resistance phenotypes of aeromonads were examined in samples from drinking and waste water treatment plants (surface, ground and disinfected water in a drinking water treatment plant, and raw and treated waste water) and tap water. Bacteria identification and intra-species variation were determined based on the analysis of the 16S rRNA, gyrB and cpn60 gene sequences. Resistance phenotypes were determined using the disc diffusion method.Aeromonas veronii prevailed in raw surface water, Aeromonas hydrophyla in ozonated water, and Aeromonas media and Aeromonas puntacta in waste water. No aeromonads were detected in ground water, after the chlorination tank or in tap water. Resistance to ceftazidime or meropenem was detected in isolates from the drinking water treatment plant and waste water isolates were intrinsically resistant to nalidixic acid. Most of the times, quinolone resistance was associated with the gyrA mutation in serine 83. The gene qnrS, but not the genes qnrA, B, C, D or qepA, was detected in both surface and waste water isolates. The gene aac(6')-ib-cr was detected in different waste water strains isolated in the presence of ciprofloxacin. Both quinolone resistance genes were detected only in the species A. media. This is the first study tracking antimicrobial resistance in aeromonads in drinking, tap and waste water and the importance of these bacteria as vectors of resistance in aquatic environments is discussed. © 2011 Elsevier Ltd.
The vaginal microbiome plays an important role in maternal and neonatal health. Imbalances in this microbiota (dysbiosis) during pregnancy are associated with negative reproductive outcomes, such as pregnancy loss and preterm birth, but the underlying mechanisms remain poorly understood. Consequently a comprehensive understanding of the baseline microbiome in healthy pregnancy is needed. We characterized the vaginal microbiomes of healthy pregnant women at 11–16 weeks of gestational age (n = 182) and compared them to those of non-pregnant women (n = 310). Profiles were created by pyrosequencing of the cpn60 universal target region. Microbiome profiles of pregnant women clustered into six Community State Types: I, II, III, IVC, IVD and V. Overall microbiome profiles could not be distinguished based on pregnancy status. However, the vaginal microbiomes of women with healthy ongoing pregnancies had lower richness and diversity, lower prevalence of Mycoplasma and Ureaplasma and higher bacterial load when compared to non-pregnant women. Lactobacillus abundance was also greater in the microbiomes of pregnant women with Lactobacillus-dominated CSTs in comparison with non-pregnant women. This study provides further information regarding characteristics of the vaginal microbiome of low-risk pregnant women, providing a baseline for forthcoming studies investigating the diagnostic potential of the microbiome for prediction of adverse pregnancy outcomes.
Data from four recent studies (S. H. Goh et al., J. Clin. Microbiol. 36:2164-2166, 1998; S. H. Goh et al., J. Clin. Microbiol. 34:818-823, 1996; S. H. Goh et al., J. Clin. Microbiol. 35:3116-3121, 1997; A. Y. C. Kwok et al., Int. J. Syst. Bacteriol. 49:1181-1192, 1999) suggest that an approximately 600-bp region of the chaperonin 60 (Cpn60) gene, amplified by PCR with a single pair of degenerate primers, has utility as a potentially universal target for bacterial identification (ID). This Cpn60 gene ID method correctly identified isolates representative of numerous staphylococcal species and Streptococcus iniae, a human and animal pathogen. We report herein that this method enabled us to distinguish clearly between 17 Enterococcus species (Enterococcus asini, Enterococcus rattus, Enterococcus dispar, Enterococcus gallinarum, Enterococcus hirae, Enterococcus durans, Enterococcus cecorum, Enterococcus faecalis, Enterococcus mundtii, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus malodoratus, Enterococcus raffinosus, Enterococcus avium, Enterococcus pseudoavium, Enterococcus new sp. strain Facklam, and Enterococcus saccharolyticus), and Vagococcus fluvialis, Lactococcus lactis, and Lactococcus garvieae. From 123 blind-tested samples, only two discrepancies were observed between the Facklam and Collins phenotyping method (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731-734, 1989) and the Cpn60 ID method. In each case, the discrepancies were resolved in favor of the Cpn60 ID method. The species distributions of the 123 blind-tested isolates were Enterococcus new sp. strain Facklam (ATCC 700913), 3; E. asini, 1; E. rattus, 4; E. dispar, 2; E. gallinarum, 20; E. hirae, 9; E. durans, 9; E. faecalis, 12; E. mundtii, 3; E. casseliflavus, 8; E. faecium, 25; E. malodoratus, 3; E. raffinosus, 8; E. avium, 4; E. pseudoavium, 1; an unknown Enterococcus clinical isolate, sp. strain R871; Vagococcus fluvialis, 4; Lactococcus garvieae, 3; Lactococcus lactis, 3; Leuconostoc sp., 1; and Pediococcus sp., 1. The Cpn60 gene ID method, coupled with reverse checkerboard hybridization, is an effective method for the identification of Enterococcus and related organisms.
The taxonomic status of Paralactobacillus selangorensis is described and, based on evidence presented, transfer of the species to the genus Lactobacillus with the name Lactobacillus selangorensis comb. nov. is proposed. This reclassification is supported by multilocus sequence analysis of the 16S rRNA gene and portions of the cpn60, pheS and rpoA genes. Mode of cell division and existing phenotypic information also show that P. selangorensis cannot be differentiated from the genus Lactobacillus. The type strain of Lactobacillus selangorensis comb. nov. is ATCC BAA-66(T) (=LMG 17710(T) =CIP 106482(T)).
Hedberg, M. E., et al. (2013). "Prevotella jejuni sp. nov., isolated from the small intestine of a child with coeliac disease." Int J Syst Evol Microbiol 63(Pt 11): 4218-4223.
Five obligately anaerobic, Gram-stain-negative, saccharolytic and proteolytic, non-spore-forming bacilli (strains CD3 : 27, CD3 : 28(T), CD3 : 33, CD3 : 32 and CD3 : 34) are described. All five strains were isolated from the small intestine of a female child with coeliac disease. Cells of the five strains were short rods or coccoid cells with longer filamentous forms seen sporadically. The organisms produced acetic acid and succinic acid as major metabolic end products. Phylogenetic analysis based on comparative 16S rRNA gene sequence analysis revealed close relationships between CD3 : 27, CD3 : 28(T) and CD3 : 33, between CD3 : 32 and Prevotella histicola CCUG 55407(T), and between CD3 : 34 and Prevotella melaninogenica CCUG 4944B(T). Strains CD3 : 27, CD3 : 28(T) and CD3 : 33 were clearly different from all recognized species within the genus Prevotella and related most closely to but distinct from P. melaninogenica. Based on 16S rRNA, RNA polymerase beta-subunit (rpoB) and 60 kDa chaperonin protein subunit (cpn60) gene sequencing, and phenotypic, chemical and biochemical properties, strains CD3 : 27, CD3 : 28(T) and CD3 : 33 are considered to represent a novel species within the genus Prevotella, for which the name Prevotella jejuni sp. nov. is proposed. Strain CD3 : 28(T) ( = CCUG 60371(T) = DSM 26989(T)) is the type strain of the proposed novel species. All five strains were able to form homologous aggregates, in which tube-like structures were connecting individual bacteria cells. The five strains were able to bind to human intestinal carcinoma cell lines at 37 degrees C.
Mycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosis competitively inhibits the nonopsonic binding of whole M. tuberculosis bacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosis capsular proteins mediate a major part of this inhibition. Two-dimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosis bacilli with macrophages. We found that recombinant Cpn60.2 can inhibit ∼57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab′)2 fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of ∼34% was seen for anti-Cpn60.2 F(ab′)2, while anti-DnaK F(ab′)2 did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosis displays both surface-associated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
CD43 is a large sialylated glycoprotein found on the surface of haematopoietic cells and has been previously shown to be necessary for efficient macrophage binding and immunological responsiveness to Mycobacterium tuberculosis. Using capsular material from M. tuberculosis and recombinant CD43-Fc, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70), bind to CD43. Competitive inhibition using recombinant protein and polyclonal F(ab')2 antibody-mediated epitope masking studies were used to evaluate M. tuberculosis binding to CD43+/+ versus CD43-/- macrophages. Results showed that Cpn60.2, but not DnaK, acts as a CD43-dependent mycobacterial adhesin for macrophage binding. Assessment of the specific binding between Cpn60.2 and CD43 showed it to be saturable, with a comparatively weak affinity in the low micromolar range. We have also shown that the ability of Cpn60.2 to competitively inhibit M. tuberculosis binding to macrophages is shared by the Escherichia coli homologue, GroEL, but not by the mouse and human Hsp60 homologues. These findings add to a growing field of research that implicates molecular chaperones as having extracellular functions, including bacterial adherence to host cells. Thus, CD43 may act as a Pattern Recognition Receptor (PRR) for bacterial homologues of the 60kDa molecular chaperone. © 2010 Blackwell Publishing Ltd.
A robust method for the identification of Campylobacter spp. based on direct sequencing of PCR-amplified partial cpn60 sequences and comparison of these to a reference database of cpn60 sequences is reported. A total of 53 Campylobacter isolates, representing 15 species, were identified and distinguished from phenotypically similar Helicobacter and Arcobacter strains. Pairwise cpn60 sequence identities between Campylobacter spp. ranged from 71 to 92%, with most between 71 and 79%, making discrimination of these species obvious. The method described overcomes limitations of existing PCR-based methods, which require time-consuming and complex post-amplification steps such as the cloning of amplification products. The results of this study demonstrate the potential for use of the reference chaperonin sequence database, cpnDB, as a tool for identification of bacterial isolates based on cpn60 sequences amplified with universal primers. © 2006 Government of Canada.
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains [~]2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca
Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.
BACKGROUND: The current recommended protocol for chaperonin-60 (cpn60) universal target based microbiome profiling includes universal PCR of microbiome samples across an annealing temperature gradient to maximize the diversity of sequences amplified. However, the value of including this gradient approach has not been formally evaluated since the optimization of a modified universal PCR primer cocktail for cpn60 PCR. PCR conditions that maximize representation of the microbiome while minimizing PCR-associated distortion of the community structure, especially in samples containing large amounts of host genomic DNA are critical. The goal of this study was to measure the effects of PCR annealing temperature and the ratio of host to bacterial DNA on the outcome of microbiota analysis, using pig microbiota as a model environment. FINDINGS: Six samples were chosen with an anticipated range of ratios of pig to bacterial genomic DNA, and universal cpn60 PCR amplification with an annealing temperature gradient was used to create libraries for pyrosequencing, resulting in 426,477 sequences from the six samples. The sequences obtained were classified as target (cpn60) or non-target based on the percent identity of their closest match to the cpnDB reference database, and target sequences were further processed to create microbiome profiles for each sample at each annealing temperature. Annealing temperature affected the amount of PCR product generated, with more product generated at higher temperatures. Samples containing proportionally more host genomic DNA yielded more non-target reads, especially at lower annealing temperatures. However, microbiome composition for each sample across the annealing temperature gradient remained consistent at both the phylum and operational taxonomic unit levels. Although some microbial sequences were detected at only one annealing temperature, these sequences accounted for a minority of the total microbiome. CONCLUSIONS: These results indicate that PCR annealing temperature does have an affect on cpn60 based microbiome profiles, but that most of the differences are due to differences in detection of low abundance sequences. Higher annealing temperatures resulted in larger amounts of PCR product and lower amounts of non-target sequence amplification, especially in samples containing proportionally large amounts of host DNA. Taken together these results provide important information to guide decisions about experimental design for cpn60 based microbiome studies.
BACKGROUND:Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database.RESULTS:Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn60 universal target sequences (also known as GroEL or Hsp60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure.CONCLUSIONS:mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly.
* 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.
A significant proportion of bacteria express two or more chaperonin genes. Chaperonins are a group of molecular chaperones, defined by sequence similarity, required for the folding of some cellular proteins. Chaperonin monomers have a mass of c. 60 kDa, and are typically found as large protein complexes containing 14 subunits arranged in two rings. The mechanism of action of the Escherichia coli GroEL protein has been studied in great detail. It acts by binding to unfolded proteins and enabling them to fold in a protected environment where they do not interact with any other proteins. GroEL can assist the folding of many proteins of different sizes, sequences, and structures, and homologues from many different bacteria can functionally replace GroEL in E. coli. What then are the functions of multiple chaperonins? Do they provide a mechanism for cells to increase their general chaperoning ability, or have they become specialized to take on specific novel cellular roles? Here I will review the genetic, biochemical, and phylogenetic evidence that has a bearing on this question, and show that there is good evidence for at least some specificity of function in multiple chaperonin genes.
A novel Gram-negative-staining strain, designated 6.2S(T), was isolated from a soil sample and identified as a biosurfactant producer. Its taxonomic position was investigated using a polyphasic approach. The cells were non-motile, non-spore-forming rods. The organism grew optimally at 30-37 degrees C, with 0-3% (w/v) NaCl, and at pH 7.0. Based on 16S rRNA gene sequence analysis, strain 6.2S(T) was found to be a member of the genus Sphingobacterium and was most closely related to four type species of the genus, showing sequence similarities of 96.8-98.9%. Partial chaperonin 60 (cpn60) gene sequence analysis was useful in resolving the phylogenetic relationships between strain 6.2S(T) and closely related taxa, with similarities ranging from 85.5% (with Sphingobacterium thalpophilum DSM 11723(T)) to 90.3% (with Sphingobacterium canadense CR11(T) and Sphingobacterium multivorum JCM 21156(T)). The results of DNA-DNA hybridization experiments between the novel strain and its closest relatives gave a DNA-DNA relatedness value of less than 70%, and consequently confirmed that this new strain did not belong to a previously described species of the genus Sphingobacterium. The major fatty acids were summed feature 3 (iso-C(15:0) 2 OH and/or C(16:1)omega7c); iso-C(15:0); iso-C(17:0) 3-OH and C(16:0). The G+C content of the genomic DNA was 40.0 mol%. According to its phenotypic and genotypic characteristics and the phylogenetic data, strain 6.2S(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium detergens sp. nov. is proposed. The type strain is 6.2S(T) ( = CECT 7938(T) = LMG 26465(T)).
Microbial populations in the gastrointestinal tract contribute to host health and nutrition. Although gut microbial ecology is well studied in livestock and domestic animals, little is known of the endogenous populations inhabiting primates or carnivora. We characterized microbial populations in fecal cultures from gorillas (Gorilla gorilla gorilla), common chimpanzees (Pan troglodytes), Hamadryas baboons (Papio hamadryas) and binturongs (Arctictis binturong) to compare the microbiomes associated with different gastrointestinal morphologies and different omnivorous feeding strategies. Each species was fed a distinct standardized diet for 2 weeks prior to fecal collection. All diets were formulated to reflect the species' feeding strategies in situ. Fresh fecal samples were pooled within species and used to inoculate in vitro batch cultures. Acetate, propionate, butyrate and valerate were measured after 24 h of incubation. Eubacterial DNA was extracted from individual fecal samples, pooled, and the cpn60 gene region was amplified and then sequenced to identify the major eubacterial constituents associated with each host species. Short chain fatty acids (P < 0.001) and methane (P < 0.001) were significantly different across species. Eubacterial profiles were consistent with fermentation data and suggest an increase in diversity with dietary fiber.
Bacteria were isolated from roots of sugarcane varieties grown in the fields of Punjab. They were identified by using API20E/NE bacterial identification kits and from sequences of 16S rRNA and amplicons of the cpn60 gene. The majority of bacteria were found to belong to the genera of Enterobacter, Pseudomonas, and Klebsiella, but members of genera Azospirillum, Rhizobium, Rahnella, Delftia, Caulobacter, Pannonibacter, Xanthomonas, and Stenotrophomonas were also found. The community, however, was dominated by members of the Pseudomonadaceae and Enterobacteriaceae, as representatives of these genera were found in samples from every variety and location examined. All isolates were tested for the presence of five enzymes and seven factors known to be associated with plant growth promotion. Ten isolates showed lipase activity and eight were positive for protease activity. Cellulase, chitinase, and pectinase were not detected in any strain. Nine strains showed nitrogen fixing ability (acetylene reduction assay) and 26 were capable of solubilizing phosphate. In the presence of 100 mg/l tryptophan, all strains except one produced indole acetic acid in the growth medium. All isolates were positive for ACC deaminase activity. Six strains produced homoserine lactones and three produced HCN and hexamate type siderophores. One isolate was capable of inhibiting the growth of 24 pathogenic fungal strains of Colletotrichum, Fusarium, Pythium, and Rhizoctonia spp. In tests of their abilities to grow under a range of temperature, pH, and NaCl concentrations, all isolates grew well on plates with 3% NaCl and most of them grew well at 4 to 41° C and at pH 11.
Phytoplasma classification established using 16S ribosomal groups and 'Candidatus Phytoplasma' taxon are mainly based on the 16S rDNA properties and do not always provide molecular distinction of the closely related strains such as those in the aster yellows group (16SrI or 'Candidatus Phytoplasma asteris'-related strains). Moreover, because of the highly conserved nature of the 16S rRNA gene, and of the not uncommon presence of 16S rDNA interoperon sequence heterogeneity, more variable single copy genes, such as ribosomal protein (rp), secY and tuf, were shown to be suitable for differentiation of closely related phytoplasma strains. Specific amplification of fragments containing phytoplasma groEL allowed studying its variability in 27 'Candidatus Phytoplasma asteris'-related strains belonging to different 16SrI subgroups, of which 11 strains were not studied before and 8 more were not studied on other genes than 16S rDNA. The restriction fragment length polymorphism (RFLP) analyses of the amplified fragments confirmed differentiation among 16SrI-A, I-B, I-C, I-F and I-P subgroups, and showed further differentiation in strains assigned to 16SrI-A, 16SrI-B and 16SrI-C subgroups. However, analyses of groEL gene failed to discriminate strains in subgroups 16SrI-L and 16SrI-M (described on the basis of 16S rDNA interoperon sequence heterogeneity) from strains in subgroup 16SrI-B. On the contrary, the 16SrI unclassified strain ca2006/5 from carrot (showing interoperon sequence heterogeneity) was differentiable on both rp and groEL genes from the strains in subgroup 16SrI-B. These results indicate that interoperon sequence heterogeneity of strains AY2192, PRIVA (16SrI-L), AVUT (16SrI-M) and ca2006/5 resulted in multigenic changes - one evolutionary step further - only in the latter case. Phylogenetic analyses carried out on groEL are in agreement with 16Sr, rp and secY based phylogenies, and confirmed the differentiation obtained by RFLP analyses on groEL amplicons. © 2011 The Authors. Annals of Applied Biology © 2011 Association of Applied Biologists.
The use of DNA-based molecular detection tools for bacterial diagnostics is hampered by the inability to distinguish signals originating from live and dead cells. The detection of live cells is typically most relevant in molecular diagnostics. DNA-intercalating dyes like ethidium monoazide and propidium monoazide (PMA) offer a possibility to selectively remove cells with compromised cell membranes from the analysis. Once these dyes enter a cell, they bind to DNA and can be covalently crosslinked to it by light exposure. PCR amplification of such modified DNA is strongly inhibited. In this study we evaluated the suitability of propidium monoazide treatment to exclude isopropanol-killed cells from detection in defined mixtures using diagnostic microarray technology. The organisms comprised Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Serratia marcescens, and Escherichia coli O157:H7. PCR products obtained from amplification of chaperonin 60 genes (cpn60; coding for GroEL) were hybridized to a custom-designed microarray containing strain-specific cpn60-based 35-mer oligonucleotide probes. Results were compared with data from quantitative PCR, which confirmed that PMA could successfully inhibit amplification of DNA from killed cells in the mixtures. Although microarray data based on analysis of end-point PCR amplicons is not quantitative, results showed a significant signal reduction when targeting killed cells and consistently agreed with qPCR results. Treatment of samples with PMA in combination with diagnostic microarray detection can therefore be considered beneficial when analyzing mixtures of intact and membrane-compromised cells. Minimization of detection signals deriving from dead cells will render data more relevant in studies including pathogen risk assessment. © 2008 Elsevier B.V. All rights reserved.
<p>Bacterial vaginosis (BV), characterized by a shift of the vaginal microbiota from a <italic>Lactobacillus</italic>-dominated community to a dense biofilm containing a complex mixture of organisms, is an important risk factor in poor reproductive health outcomes. The Nugent score, based on Gram stain, is used to diagnose BV and <italic>Gardnerella vaginalis</italic> abundance in the sample is one factor determining Nugent score. A high Nugent score is indicative of BV but does not always correspond to the presence of clinical symptoms. <italic>G. vaginalis</italic> is recognized as a heterogeneous group of organisms, which can also be part of the normal, healthy vaginal microbiome. In addition, asymptomatic BV and non-<italic>Gardnerella</italic> types of BV are being recognized. In an attempt to resolve the heterogeneous group of <italic>G. vaginalis</italic>, a phylogenetic tree of <italic>cpn</italic>60 universal target sequences from <italic>G. vaginalis</italic> isolates was constructed that indicates the existence of four subgroups of <italic>G. vaginalis</italic>. This subdivision, supported by whole genome similarity calculation of representative strains using JSpecies, demonstrates that these subgroups may represent different species. The <italic>cpn</italic>60 subgroupings did not correspond with the Piot biotyping scheme, but did show consistency with ARDRA genotyping and sialidase gene presence. Isolates from all four subgroups produced biofilm <italic>in vitro</italic>. We also investigated the distribution of <italic>G. vaginalis</italic> subgroups in vaginal samples from Kenyan women with Nugent scores consistent with BV, Intermediate and Normal microbiota (n = 44). All subgroups of <italic>G. vaginalis</italic> were detected in these women, with a significant difference (z = −3.372, n = 39, p = 0.001) in frequency of <italic>G. vaginalis</italic> subgroup B between BV and Normal groups. Establishment of a quantifiable relationship between <italic>G. vaginalis</italic> subgroup distribution and clinical status could have significant diagnostic implications.</p>
Samples of a wild grass showing white leaf symptoms were collected in 2015 in Veracruz, Mexico around sugarcane plantations. DNA amplification, sequencing and phylogenetic analysis revealed the presence of a ‘Candidatus Phytoplasma asteris’-related strain in the plants that were positive using 16S-based and cpn60 PCR assays. The strain determined to be associated with the grass white leaf disease was identified as a member of the 16SrI-B subgroup and a member of the cpn60 I-IIIB subgroup through in silico RFLP analysis. This is the first report of phytoplasma-infected grass plants associated with white leaf disease in Mexico. The implications of these findings are vital for the management of other plant hosts of the family Poaceae such as sugarcane and corn, and point to weedy grasses as a potential source of phytoplasma inoculum for nearby crop plants.
Catharantus roseus, known as periwinkle, is a plant highly susceptible to phytoplasma infection. Periwinkle plants showing proliferation symptoms were detected during 2013-2014 in four geographically distant states in Mexico. The presence of phytoplasma was confirmed through the amplification of the 16S F2nR2 and the cpn60 UT sequences from symptomatic plants. The sequencing, phylogenetic analysis, and in vitro RFLP revealed that the isolates were ‘Candidatus Phytoplasma asteris’– related strains and members of the 16SrI-B subgroup, confirming for first time the association of this phytoplasma group and the Mexican periwinkle proliferation disease. In this study we demonstrated that the use of the approximately 550 pb cpn60 universal target sequences allowed the differentiation of two different 16SrI-B strains designated here MePP-Centre, and MePP-South.
In the 2013–2014 growing season, field surveys were conducted in native corn fields located in high altitude agricultural communities in the ‘Sierra Norte de Puebla’ in Mexico. Symptoms typical of maize bushy stunt (MBS) disease were observed and DNA extracted from symptomatic native corn plants was used as template to confirm the presence of phytoplasmas. Amplification and sequencing of 16S rRNA-encoding sequences and chaperonin 60 universal target (cpn60 UT) sequences followed by in vitro restriction fragment length polymorphism and phylogenetic analyses revealed that the phytoplasma detected belongs to the subgroup 16SrI-B, ‘Candidatus Phytoplasma asteris’. Based on 16S rRNA-encoding gene sequence analysis and on a single nucleotide polymorphism within the cpn60 UT sequence, two MBS strains, MBS-Puebla and MBS-Veracruz, were identified. This is the first detection of MBS phytoplasma (MBSP) affecting native corn and the first molecular survey made in corn fields in Mexico to detect and characterize MBSP. We discuss these results in light of the potential evolutionary relationship between corn and MBSP.
Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma. A fluorescent microsphere hybridization assay was designed that was capable of detecting SbGP/MPV phytoplasma in infected plant tissues, successfully differentiating it from other known phytoplasma cpn60 UT sequences, while identifying a double infection with SbGP/MPV and aster yellows (16SrI) phytoplasma. Two quantitative assays, quantitative real-time PCR (qRT-PCR) and droplet digital PCR (ddPCR), gave similar results in infected samples. Finally, a loop-mediated isothermal amplification (LAMP) assay provided rapid detection of SbGP/MPV phytoplasma DNA. Application of these assays revealed that SbGP/MPV phytoplasma is widely distributed in Central Mexico, with positive samples identified from eleven localities within three states separated by hundreds of kilometres. These results also provide tools for determining the presence and geographic distribution of this pathogen in plant and insect samples in other localities.
<p>Whereas the infant gut microbiome is the subject of intense study, relatively little is known regarding the nares microbiome in newborns and during early life. This study aimed to survey the typical composition and diversity of human anterior nare microflora for developing infants over time, and to explore how these correlate to their primary caregivers. Single nare swabs were collected at five time points over a one-year period for each subject from infant-caregiver pairs. Our study comprised of 50 infants (recruited at 2 weeks, post delivery) and their 50 primary caregivers. Applying the chaperonin-60 (<italic>cpn60)</italic> universal target (UT) amplicon as our molecular barcoding marker to census survey the microbial communities, we longitudinally surveyed infant nares microbiota at 5 time points over the course of the first year of life. The inter- and intra-subject diversity was catalogued and compared, both longitudinally and relative to their adult primary caregivers. Although within-subject variability over time and inter-subject variability were both observed, the assessment detected only one or two predominant genera for individual infant samples, belonging mainly to phyla Actinobacteria, Firmicutes, and Proteobacteria. Consistent with previously observed microbial population dynamics in other body sites, the diversity of nares microflora increased over the first year of life and infants showed differential operational taxonomic units (OTUs) relative to their matched primary caregiver. The collected evidence also support that both temporal and seasonal changes occur with respect to carriage of potentially pathogenic bacteria (PPBs), which may influence host predisposition to infection. This pilot study surveying paired infant/caregiver nare microbiomes provides novel longitudinal diversity information that is pertinent to better understanding nare microbiome development in infants.</p>
Pratt, D. L., et al. (2012). "Influence of mass burial of animal carcasses on the types and quantities of microorganisms within a burial site." Transactions of the ASABE 55(6): 2195-2212.
Microbial communities were characterized through a depth of 10 m in and near an eight-year-old burial site that was implemented as a control measure for cases of chronic wasting disease among wild elk. Quantitative PCR based on the 16S rRNA-encoding gene showed that the burial trench had significantly (2 to 5 orders of magnitude) more bacterial 16S rRNA-encoding genes g-1 soil within and up to 2 m below the burial site compared to a nonburial control core sample at the same depths. Topsoil and depths below the burial site (>6 m) showed similar quantities of 16S rRNA genes for both cores. Furthermore, when microbial communities were examined by cpn60 universal target amplification and pyrosequencing, 5825 operational taxonomic units (OTU) were found at a variety of abundances in all of the 13 core samples that were analyzed. Taxonomic analysis indicated that the overall community composition changed considerably with increasing depth, and that the burial core community was distinct from that of the control core at all depths. Organisms associated with phosphate accumulation, nitrogen fixation, and ammonium oxidation were found in highest abundance near the surface of the burial core (up to 2.5 m), while organisms associated with ammonification were found at the burial depth, consistent with an increase in ammonium concentration in pore water. Sequences from organisms associated with dissimilatory metal reduction were concentrated just below the burial depth (4.5 to 5.5 m). Anaerobic microorganisms dominated the microbial community at the burial site (3.75 m). The approaches described in this study provided distinct sequences that can be used to develop molecular assays for organism tracking and overall fingerprinting of microbes associated with burial sites. These data can be helpful in site evaluation and may help in the selection of future burial sites. © 2012 American Society of Agricultural and Biological Engineers.
Two strains of aerobic, non-spore-forming, Gram-negative, rod-shaped bacteria (ND5 and MY14T), previously isolated from urban soil using the membrane-filter enrichment technique, were characterized. Analysis of their 16S rRNA gene sequence grouped strains ND5 and MY14T within the family Oxalobacteraceae (Betaproteobacteria). The highest pairwise sequence similarities for strain ND5 were found with members of the genus Herminiimonas, namely with Herminiimonas saxobsidens NS11T (99.8%) and Herminiimonas glaciei UMB49T (99.6%). Although some fatty acid profiles, physiological and biochemical differences exist between strain ND5 and the respective Herminiimonas-type strains, DNA-DNA hybridization experiments confirm that strain ND5 is a member of the H. glaciei genospecies. Taxonomical analyses revealed a wider range of variability within this genus than considered previously. The highest pairwise nucleotide similarity for strain MY14 T was found with Oxalicibacterium flavum (96.8%). Phylogenetic analyses based on 16S rRNA and cpn60 gene sequences, DNA-DNA hybridization, fatty acid profiles, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MY14T from other Oxalicibacterium species representing a new species, for which the name Oxalicibacterium solurbis sp. nov. (type strain MY14T=NBRC 102665T,=CCM 7664T) is proposed. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.
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.
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.
A pink-pigmented, facultatively methylotrophic bacterium, strain 23e(T), was isolated from the leaves of Gnaphalium spicatum (cudweed). The cells of strain 23e(T) were Gram-reaction negative, motile and non-spore-forming rods. On the basis of 16S rRNA gene sequence similarities, strain 23e(T) was related to Methylobacterium organophilum ATCC 27886(T) (97.1%) and Methylobacterium marchantiae JT1(T) (97%), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 97%. Major cellular fatty acids were C(18:1)omega7c, C(16:00) and C(18:0). The results of DNA-DNA hybridization, phylogenetic analyses based on 16S rRNA and cpn60 gene sequences, fatty acid profiles, whole-cell matrix-assisted laser desorption/ionization time of flight/MS analysis, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 23e(T) from the phylogenetically closest relatives. We propose that strain 23e(T) represents a novel species within the genus Methylobacterium, for which the name Methylobacterium gnaphalii sp. nov. is proposed. The type strain is 23e(T) (=DSM 24027(T)=NBRC 107716(T)).
Genus Xanthomonas comprises many economically important plant pathogens that affect a wide range of hosts. Indeed, fourteen Xanthomonas species/pathovars have been regarded as official quarantine bacteria for imports in China. To date, however, a rapid and accurate method capable of identifying all of the quarantine species/pathovars has yet to be developed. In this study, we therefore evaluated the capacity of DNA barcoding as a digital identification method for discriminating quarantine species/pathovars of Xanthomonas. For these analyses, 327 isolates, representing 45 Xanthomonas species/pathovars, as well as five additional species/pathovars from GenBank (50 species/pathovars total), were utilized to test the efficacy of four DNA barcode candidate genes (16S rRNA gene, cpn60, gyrB, and avrBs2). Of these candidate genes, cpn60 displayed the highest rate of PCR amplification and sequencing success. The tree-building (Neighbor-joining), 'best close match', and barcode gap methods were subsequently employed to assess the species- and pathovar-level resolution of each gene. Notably, all isolates of each quarantine species/pathovars formed a monophyletic group in the neighbor-joining tree constructed using the cpn60 sequences. Moreover, cpn60 also demonstrated the most satisfactory results in both barcoding gap analysis and the 'best close match' test. Thus, compared with the other markers tested, cpn60 proved to be a powerful DNA barcode, providing a reliable and effective means for the species- and pathovar-level identification of the quarantine plant pathogen Xanthomonas.
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 noncompetitive 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.
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 4.2 kb 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.
Characterization of complex microbial communities is frequently based on the examination of polymerase chain reaction amplified sequences from a single phylogenetic marker, usually the 16S rRNA gene. However, this commonly used target often does not offer robust resolution of species or sub-species and is thus not a sufficiently informative target for understanding microbial population dynamics occurring at the strain level. We have used the cpn60 universal target sequence to characterize Enterococcus isolates from feces of growing pigs and have shown that sub-species groups, not detected using 16S rRNA sequences, can be resolved. Furthermore, groups resolved by cpn60-based phylogenetic analysis have distinct phenotypes. We report changes in the structure and function of Enterococcus communities in pig feces sampled from individual animals at three times, from suckling through to maturity. Enterococcus faecalis was largely replaced by Enterococcus hirae between suckling and 9 weeks of age, and a shift from one sub-species group of E. hirae to another was observed in all animals between 9 and 15 weeks. Conversely, E. faecalis strains remained consistent throughout the study period. Our results demonstrate that cpn60 sequences can be used to detect strain level changes in Enterococcus populations during succession in the fecal microbiota of growing pigs. © Springer Science + Business Media, LLC 2009.
