Characterization of Neutrophilic Fe(II)-Oxidizing Bacteria Isolated from the Rhizosphere of Wetland Plants and Description of Ferritrophicum radicicola gen. nov. sp. nov., and Sideroxydans paludicola sp. nov.

Iron deposits (Fe plaque) on wetland plant roots contain abundant microbial populations, including Fe(II)-oxidizing bacteria (FeOB) that have not been cultured previously. In this study, 4 strains of Fe plaque-associated FeOB were isolated from 4 species of wetland plants. All 4 isolates grew in tight association with Fe-oxides, but did not form any identifiable Fe-oxide structures. All strains were obligate lithotrophic Fe(II)-oxidizers that were microaerobic, and were unable to use other inorganic or organic energy sources. One strain, BrT, was shown to fix ¹⁴ CO ₂ at a rate consistent with its requirement for total cell carbon. The doubling times for the strains varied between 9.5 and 15.8 hours. The fatty acid methyl ester (FAME) profiles of 2 strains, BrT and CCJ, revealed that 16:0, 15:1 isoG, and 14:0 were dominant fatty acids. Phylogenetic analysis of the 16S rRNA gene indicated that all the strains were Betaproteobacteria. Two of the strains, BrT and Br-1 belong to a new species, Sideroxydans paludicola; a third strain, LD-1, is related to Sideroxydans lithotrophicus, a recently described species of FeOB. The fourth isolate, Ferritrophicum radicicola, represented a new genus in a new order of Betaproteobacteria, the Ferritrophicales. There are no other cultured isolates in this order. A small subunit rRNA gene-based, cultivation-independent analysis of Typha latifolia collected from a wetland revealed terminal restriction fragment profiles (tRFLP) consistent with the presence of these bacteria in the rhizosphere. These novel organisms likely play an important role in Fe(II) oxidation kinetics and Fe cycling within many terrestrial and freshwater environments.     

Phylogenetic diversity of termite gut spirochaetes

A molecular phylogenetic analysis was done of not-yet-cultured spirochaetes inhabiting the gut of the termite, Reticulitermes flavipes (Kollar). Ninety-eight clones of near-full-length spirochaetal 16S rDNA genes were classified by ARDRA pattern and by partial sequencing. All clones grouped within the genus Treponema , and at least 21 new species of Treponema were recognized within R. flavipes alone. Analysis of 190 additional clones from guts of Coptotermes formosanus Shiraki and Zootermopsis angusticollis (Hagen), as well as published data on clones from Cryptotermes domesticus (Haviland), Mastotermes darwiniensis Froggatt, Nasutitermes lujae (Wasmann) and Reticulitermes speratus (Kolbe), revealed a similar level of novel treponemal phylogenetic diversity in these representatives of five of the seven termite families. None of the clones was closely related (i.e. all bore ≤ 91% sequence similarity) to any previously recognized treponeme. The data also revealed the existence of two major phylogenetic groups of treponemes: one containing all of the currently known isolates of Treponema and a large number of phylotypes from the human gingival crevice, but only a minority of the termite gut spirochaete clones; another containing the majority of termite spirochaete clones and two Spirochaeta ( S. caldaria and S. stenostrepta ), which, although free living, group within the genus Treponema on the basis of 16S rRNA sequence. Signature nucleotides that almost perfectly distinguished the latter group, herein referred to as the 'termite cluster', occurred at the following ( E. coli numbering) positions: 289-G · C-311; A at 812; and an inserted nucleotide at 1273. The emerging picture is that the long-recognized and striking morphological diversity of termite gut spirochaetes is paralleled by their phylogenetic diversity and may reflect substantial physiological diversity as well.     

The RDP-II (Ribosomal Database Project)

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [Nucleic Acids Res. (2000), 28, 173-174], continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 8.0 (June 1, 2000) consisted of 16 277 aligned prokaryotic small subunit (SSU) rRNA sequences while the number of eukaryotic and mitochondrial SSU rRNA sequences in aligned form remained at 2055 and 1503, respectively. The number of prokaryotic SSU rRNA sequences more than doubled from the previous release 14 months earlier, and approximately 75% are longer than 899 bp. An RDP-II mirror site in Japan is now available (http://wdcm.nig.ac.jp/RDP/html/index.h tml). RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment polymorphism experiments. The RDP-II email address for questions and comments has been changed from curator@cme.msu.edu to rdpstaff@msu.edu.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Computational aspects of systematic biology

We review the resources available to systematic biologists who wish to use computers to build classifications. Algorithm development is in an early stage, and only a few examples of integrated applications for systematic biology are available. The availability of data is crucial if systematic biology is to enter the computer age.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Genomic Encyclopedia of Type Strains,Phase I: The one thousand microbial genomes (KMG-I) project

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project with the objective of sequencing 250 bacterial and archaeal genomes. The two major goals of that project were (a) to test the hypothesis that there are many benefits to the use the phylogenetic diversity of organisms in the tree of life as a primary criterion for generating their genome sequence and (b) to develop the necessary framework, technology and organization for large-scale sequencing of microbial isolate genomes. While the GEBA pilot project has not yet been entirely completed, both of the original goals have already been successfully accomplished, leading the way for the next phase of the project.Here we propose taking the GEBA project to the next level, by generating high quality draft genomes for 1,000 bacterial and archaeal strains. This represents a combined 16-fold increase in both scale and speed as compared to the GEBA pilot project (250 isolate genomes in 4+ years). We will follow a similar approach for organism selection and sequencing prioritization as was done for the GEBA pilot project (i.e. phylogenetic novelty, availability and growth of cultures of type strains and DNA extraction capability), focusing on type strains as this ensures reproducibility of our results and provides the strongest linkage between genome sequences and other knowledge about each strain. In turn, this project will constitute a pilot phase of a larger effort that will target the genome sequences of all available type strains of the Bacteria and Archaea.     

Interrelations between the Microbiotas in the Litter and in the Intestines of Commercial Broiler Chickens

The intestinal microbiota of broiler chickens and the microbiota in the litter have been well studied, but the interactions between these two microbiotas remain to be determined. Therefore, we examined their reciprocal effects by analyzing the intestinal microbiotas of broilers reared on fresh pine shavings versus reused litter, as well as the litter microbiota over a 6-week cycle. Composite ileal mucosal and cecal luminal samples from birds (n = 10) reared with both litter conditions (fresh versus reused) were collected at 7, 14, 21, and 42 days of age. Litter samples were also collected at days 7, 14, 21, and 42. The microbiotas were profiled and compared within sample types based on litter condition using PCR and denaturing gradient gel electrophoresis (PCR-DGGE). The microbiotas were further analyzed using 16S rRNA gene clone libraries constructed from microbiota DNA extracted from both chick intestinal and litter samples collected at day 7. Results showed significant reciprocal effects between the microbiotas present in the litter and those in the intestines of broilers. Fresh litter had more environmental bacteria, while reused litter contained more bacteria of intestinal origin. Lactobacillus spp. dominated the ileal mucosal microbiota of fresh-litter chicks, while a group of bacteria yet to be classified within Clostridiales dominated in the ileal mucosal microbiota in the reused-litter chicks. The Litter condition (fresh versus reused) seemed to have a more profound impact on the ileal microbiota than on the cecal microbiota. The data suggest that the influence of fresh litter on ileal microbiota decreased as broilers grew, compared with temporal changes observed under reused-litter rearing conditions.The intestines of broiler chickens harbor a complex microbiota that plays an important role in the growth and health of the bird. Nurmi and Rantala (27) provided the first reported evidence that a healthy gut microbiota could protect broiler chicks against a challenge by enteric pathogens. That study led to the concept of “competitive exclusion.” Since then, the colonization of the intestines by beneficial bacteria has been shown to promote epithelial cell turnover (34), increase mucous production (26), upregulate expression of genes involved in several important intestinal functions (14), and help with reinforcement of the mucosal barrier, modulation of the immune system, and metabolism of nutrients by the host (46). Although studies conducted using either cultivation-based or molecular biology analyses have documented that the intestinal microbiota of mature broiler chickens is relatively stable (10, 24, 45), this microbiota is still dynamic and can be manipulated to a large extent (8, 31, 37).Commercial broiler flocks in the United States are primarily floor raised in enclosed, environmentally controlled facilities. Within these commercial broiler houses, poultry litter can be reused for a year or longer if managed well and maintained in a relatively dry state (6). At an estimated accumulation rate of 1.45 metric tons per 1,000 broilers, the U.S. poultry industry, which produces >8.5 billion broilers per year (39), produces >12.3 million metric tons of poultry litter annually (4). Management and disposal of such a large quantity of poultry litter are two of the greatest challenges faced by U.S. broiler producers (6). Reuse of poultry litter can help alleviate this challenge, but there is concern that the reused-litter environment, both biotic and abiotic, may negatively affect the intestinal microbiota of the bird, potentially resulting in poor health and reduced production efficiency.Poultry litter is primarily a mixture of bedding materials and bird excreta. In addition to variation in the physiochemical properties, reused poultry litter harbors microbes of typically intestinal origin that are not commonly present in fresh litter (23, 35). During a broiler growth cycle, a constant influx of nutrients and intestinal microbes results in a complex litter microbiota. With continued reuse, the litter environment becomes more complex, which may have a profound impact on flock growth performance and health. Recognizing the importance of the litter environment, especially in commercial broiler houses with high stocking densities, several research groups have investigated how the physiochemical properties of broiler litter affect the growth and health of the birds (15) and the microbiota in the litter, using either cultivation-based or molecular biology techniques (22, 23, 33). Early studies focused on the effect of litter on enteric pathogens, including Salmonella (35, 12, 28) and cellulitis-causing Escherichia coli (38). From analyzing 16S rRNA gene clone libraries, Lu et al. (23) showed that in broiler litter at various stages of reuse, low-G+C Gram-positive bacteria predominated. Recent studies have focused on the interplay between the unique physiochemical conditions within the litter and the endogenous microbiota (22, 29), as well as the occurrence and persistence of antibiotic resistance in poultry litter (13, 17), providing some insight into the litter microbiota and the intestinal microbiota in broiler chickens. However, the relationship between litter and intestinal microbiotas has not been investigated. Since chickens consume some litter materials, we hypothesized that the intestinal and litter microbiotas may affect each other with respect to their composition and diversity. Using PCR with denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rRNA gene clone library analysis, we tested this hypothesis by examining the effect of the litter microbiota on the ontogeny of the intestinal microbiota in broilers over a 6-week growth cycle.     

Use of bioluminescence imaging to monitor Campylobacter survival in chicken litter

Aim: The aim of this study was to develop a novel approach for characterizing the growth and persistence of Campylobacter in different poultry‐rearing environments. Specifically, we constructed bioluminescent Campylobacter strains and used them to monitor the survival of these pathogens in litter (bedding) material. Methods and Results: We inserted shuttle plasmids carrying the luminescence genes (luxCDABE) into C. jejuni and C. coli to construct bioluminescent strains of these pathogens. The strains were spiked into microcosms containing samples of litter‐washings and dry litter collected from different enclosures that housed broiler chickens. Our results show that C. jejuni and C. coli survived for at least 20 days in reused (old) litter while the growth of these pathogens was inhibited in clean (new) litter. Furthermore, our results suggest that the availability of nutrients and the condition of the litter (reused vs new) are important factors in the persistence of these pathogens. Conclusions: Reused litter can potentially predispose chickens to Campylobacter contamination and maintaining clean litter might reduce the incidences of colonization with these pathogens. Significance and Impact of the Study: Bioluminescence provided a simple, sensitive, and rapid approach for analysing the growth dynamics of Campylobacter. Using this technology, we highlighted the potential role of litter material in maintaining these pathogens in the chicken environment.