Batch vs. continuous direct compression - a comparison of material processability and final tablet quality.

In this study, an in-depth comparison was made between batch and continuous direct compression using similar compression set-ups. The overall material processability and final tablet quality were compared and evaluated. Correlations between material properties, process parameters and final tablet properties were made via multivariate data analyses. In total, 10 low-dosed (1% w/w) and 10 high-dosed (40% w/w) formulations were processed, using a total of 10 different fillers/filler combinations. The trials indicated that the impact of filler type, drug load or process settings was similar for batch and continuous direct compression. The main differentiator between batch and continuous was the flow dynamics in the operating system, where properties related to flow, compressibility and permeability played a crucial role. The less consistent flow throughout a batch process resulted in a significantly higher variability within the tablet press (σCF) and for the tablet quality responses (σMass, σTS). However, the better controlled blending procedure prior to batch processing was reflected in a more consistent API concentration variability. Overall, the comparison showed the benefits of selecting appropriate excipients and process settings to achieve a specific outcome, keeping in mind some key differentiators between both processes.

Modelling thermodynamic feedback on the metabolism of hydrogenotrophic methanogens.

The magnitude of the Gibbs free energy change of the substrate transformation that supports the growth of a microbe is decreased when the concentrations of the substrates are decreased and when the concentrations of the products of metabolism are increased. Microbes require a supply of ATP for cell maintenance and growth, and coupling the transformation of substrates to products with the formation of ATP also decreases the magnitude of the Gibbs free energy change. Here we include these three thermodynamic controllers (substrate and product concentration, and ATP formation) in a model of substrate transformation by hydrogenotrophic methanogens that results in a number of realistic behaviours. First, a threshold for substrate use emerges, below which the methanogen cannot metabolise its substrate. Under this model, microbes that capture more of the Gibbs free energy change from substrate transformation in the form of ATP have greater thresholds for their substrate, in line with observations of actual microbes. Second, an apparent saturation constant emerges that is controlled by the thermodynamics of the reaction. This increases with increasing ATP synthesis per substrate, so that methanogens that conserve more ATP grow faster at higher substrate concentrations, but are less competitive at low substrate concentrations. As a result, simply changing the ATP yield (moles of ATP per mole of substrate) results in methanogens with differing ecological strategies through thermodynamic impacts on their metabolism. Third, end-product inhibition through thermodynamic feedback can limit the growth of microbes, and those that capture more ATP per substrate are limited by smaller product concentrations than those that capture less ATP.

Temporal fermentation and microbial community dynamics in rumens of sheep grazing a ryegrass-based pasture offered either in the morning or in the afternoon.

Eight ruminally-fistulated wethers were used to examine the temporal effects of afternoon (PM; 1600h) v. morning (AM; 0800 h) allocation of fresh spring herbage from a perennial ryegrass (Lolium perenne L.)-based pasture on fermentation and microbial community dynamics. Herbage chemical composition was minimally affected by time of allocation, but daily mean ammonia concentrations were greater for the PM group. The 24-h pattern of ruminal fermentation (i.e. time of sampling relative to time of allocation), however, varied considerably for all fermentation variables (P⩽0.001). Most notably amongst ruminal fermentation characteristics, ammonia concentrations showed a substantial temporal variation; concentrations of ammonia were 1.7-, 2.0- and 2.2-fold greater in rumens of PM wethers at 4, 6 and 8h after allocation, respectively, compared with AM wethers. The relative abundances of archaeal and ciliate protozoal taxa were similar across allocation groups. In contrast, the relative abundances of members of the rumen bacterial community, like Prevotella 1 (P=0.04), Bacteroidales RF16 group (P=0.005) and Fibrobacter spp. (P=0.008) were greater for the AM group, whereas the relative abundance of Kandleria spp. was greater (P=0.04) for the PM group. Of these taxa, only Prevotella 1 (P=0.04) and Kandleria (P<0.001) showed a significant interaction between time of allocation and time of sampling relative to feed allocation. Relative abundances of Prevotella 1 were greater at 2h (P=0.05), 4h (P=0.003) and 6h (P=0.01) after AM allocation of new herbage, whereas relative abundances of Kandleria were greater at 2h (P=0.003) and 4h (P<0.001) after PM allocation. The early post-allocation rise in ammonia concentrations in PM rumens occurred simultaneously with sharp increases in the relative abundance of Kandleria spp. and with a decline in the relative abundance of Prevotella. All measures of fermentation and most microbial community composition data showed highly dynamic changes in concentrations and genus abundances, respectively, with substantial temporal changes occurring within the first 8h of allocating a new strip of herbage. The dynamic changes in the relative abundances of certain bacterial groups, in synchrony with a substantial diurnal variation in ammonia concentrations, has potential effects on the efficiency by which N is utilised by the grazing ruminant.

Methane emissions changed nonlinearly with graded substitution of alfalfa silage with corn silage and corn grain in the diet of sheep and relation with rumen fermentation characteristics in vivo and in vitro.

Feeding grain and corn silage have been proposed as practices to reduce enteric methane (CH) emissions per unit of intake from ruminants, but the inclusion level required in the diet is normally not specified. The objectives of the current study were to determine the CH emission factor (g/kg DMI) of sheep fed alfalfa silage substituted with increasing levels of corn silage or corn grain at a fixed DMI level (2% of BW) and determine its relationship with rumen fermentation characteristics and microbial community composition and with in vitro fermentation characteristics of the same diets incubated using a standard laboratory method. Romney ewe hoggets (approximately 14 mo old; = 64) were randomly allocated to 8 dietary treatments, which included chaffed alfalfa silage alone or substituted with either 25, 50, 75 or 100% corn silage or 25, 50 or 65% rolled corn grain on a DM basis. After acclimatization to the diet, DMI and CH emissions were measured from individual sheep for 2 consecutive days in open-circuit respiration chambers and a rumen sample was collected at 3 h after feeding. The same diets were also incubated in an automated in vitro gas production system for 48 h using rumen liquid of fistulated nonlactating dairy cows grazing pasture. Increasing the substitution of alfalfa silage with corn silage or corn grain in the diet of sheep resulted in a quadratic response ( < 0.01) in CH emissions per unit of DMI (CH/DMI) with either supplement. For both supplements, CH/DMI increased in mixtures of up to 50% supplement inclusion and then decreased with greater supplement inclusion, especially with corn grain inclusion, but the level did not fall below that for 100% alfalfa silage. The ratio of acetate + butyrate to propionate + valerate and the propionate proportion alone in rumen liquid were the strongest single predictors for CH/DMI in the overall data set and explained 37.1 and 32.5%, respectively, of the variation in CH/DMI. Methanogens of (21.1% of total methanogens; = 0.247) and (10.7% of total methanogens; = -0.411) clades had weak to moderate correlations with in vivo CH/DMI. There was a weak quadratic relationship ( < 0.35) between in vivo CH/DMI and the in vitro parameters of gas and CH production and total VFA, whereas there was a moderate relationship ( = -0.50) between in vivo CH/estimated rumen degradable carbohydrates and in vitro CH/DM. In conclusion, CH/DMI changed in a nonlinear fashion with increasing supplement inclusion in the alfalfa forage diet when fed at 2% of BW to sheep; however, implications on predicting its influence on greenhouse gas emissions per unit of animal product, for whole farm emissions in life cycle analysis or total national emissions in the national inventories, should be determined.

Sampling procedure for the measurement of dissolved hydrogen and volatile fatty acids in the rumen of dairy cows.

Dissolved hydrogen (dH) influences the pathways of VFA production and is a precursor of methane formation in the rumen. Measurements of dH in rumen fluid taken at the same time as measuring other rumen fermentation end products would improve our quantitative understanding of the role of dH as a controller of rumen fermentation. Sample collections though a rumen cannula and using oral stomach tubing were compared for measurements of dissolved gases and fermentation end products in the rumen fluid of 4 ruminally cannulated dairy cows fed a total mixed ration of corn silage and concentrate. Rumen fluid was collected at 0, 2.5, and 6 h after morning feeding through the cannula from cranial dorsal rumen, cranial ventral rumen, central rumen, caudal dorsal rumen, and caudal ventral rumen and in parallel by oral stomach tubing at 2 insertion depths of 180 cm (sampling the central rumen) and 200 cm (sampling the caudal dorsal rumen). The cranial dorsal rumen had the greatest pH and smallest VFA concentration among 5 sites sampled. Samples collected by oral stomach tubing had greater ( < 0.001) rumen pH and less ( < 0.001) dissolved methane (dCH) and lower VFA concentration than that collected through rumen cannula. The dH concentrations were positively correlated ( > 0.8) in rumen samples collected by the 2 sampling techniques, with a concordance correlation coefficient larger than 0.8 and scale shift being about 0.1 away from unity. The variations in the measurement of dH, dCH, pH, and VFA in samples collected by oral stomach tubing are most likely the result of saliva contamination. The time of sampling relative to feeding had significant influence ( < 0.01) on dissolved gases and fermentation end products, with the greatest concentrations of dH, dCH, and VFA measured 2.5 h after morning feeding. The dH was correlated positively ( > 0.58) with dCH and negatively ( < -0.65) with the estimated net H production relative to the amount of VFA produced. This indicated that greater dH enhanced rumen CH production and also led to fermentation pathways that produce less H, such as enhanced propionate and butyrate production. In summary, oral stomach tubing could be a feasible method to measure ruminal dH in intact animals, but caution should be taken to minimize saliva contamination. Measurements made using both techniques yield similar conclusions for the effects of dH on fermentation pathways and CH generation.

A modified version of the Molly rumen model to quantify methane emissions from sheep.

We modified the rumen submodel of the Molly dairy cow model to simulate the rumen of a sheep and predict its methane emissions. We introduced a rumen hydrogen (H2) pool as a dynamic variable, which (together with the microbial pool in Molly) was used to predict methane production, to facilitate future consideration of thermodynamic control of methanogenesis. The new model corrected a misspecification of the equation of microbial H2 utilization in Molly95, which could potentially give rise to unrealistic predictions under conditions of low intake rates. The new model included a function to correct biases in the estimation of net H2 production based on the default stoichiometric relationships in Molly95, with this function specified in terms of level of intake. Model parameters for H2 and methane production were fitted to experimental data that included fresh temperate forages offered to sheep at a wide range of intake levels and then tested against independent data. The new model provided reasonable estimates relative to the calibration data set, but a different parameterization was needed to improve its predicted ability relative to the validation data set. Our results indicate that, although feedback inhibition on H2 production and methanogen activity increased with feeding level, other feedback effects that vary with diet composition need to be considered in future work on modeling rumen digestion in Molly.

RUMINANT NUTRITION SYMPOSIUM: Use of genomics and transcriptomics to identify strategies to lower ruminal methanogenesis.

Globally, methane (CH4) emissions account for 40% to 45% of greenhouse gas emissions from ruminant livestock, with over 90% of these emissions arising from enteric fermentation. Reduction of carbon dioxide to CH4 is critical for efficient ruminal fermentation because it prevents the accumulation of reducing equivalents in the rumen. Methanogens exist in a symbiotic relationship with rumen protozoa and fungi and within biofilms associated with feed and the rumen wall. Genomics and transcriptomics are playing an increasingly important role in defining the ecology of ruminal methanogenesis and identifying avenues for its mitigation. Metagenomic approaches have provided information on changes in abundances as well as the species composition of the methanogen community among ruminants that vary naturally in their CH4 emissions, their feed efficiency, and their response to CH4 mitigators. Sequencing the genomes of rumen methanogens has provided insight into surface proteins that may prove useful in the development of vaccines and has allowed assembly of biochemical pathways for use in chemogenomic approaches to lowering ruminal CH4 emissions. Metagenomics and metatranscriptomic analysis of entire rumen microbial communities are providing new perspectives on how methanogens interact with other members of this ecosystem and how these relationships may be altered to reduce methanogenesis. Identification of community members that produce antimethanogen agents that either inhibit or kill methanogens could lead to the identification of new mitigation approaches. Discovery of a lytic archaeophage that specifically lyses methanogens is 1 such example. Efforts in using genomic data to alter methanogenesis have been hampered by a lack of sequence information that is specific to the microbial community of the rumen. Programs such as Hungate1000 and the Global Rumen Census are increasing the breadth and depth of our understanding of global ruminal microbial communities, steps that are key to using these tools to further define the science of ruminal methanogenesis.

Progress in the development of vaccines against rumen methanogens.

Vaccination against rumen methanogens offers a practical approach to reduce methane emissions in livestock, particularly ruminants grazing on pasture. Although successful vaccination strategies have been reported for reducing the activity of the rumen-dwelling organism Streptococcus bovis in sheep and S. bovis and Lactobacillus spp. in cattle, earlier approaches using vaccines based on whole methanogen cells to reduce methane production in sheep have produced less promising results. An anti-methanogen vaccine will need to have broad specificity against methanogens commonly found in the rumen and induce antibody in saliva resulting in delivery of sufficiently high levels of antibodies to the rumen to reduce methanogen activity. Our approach has focussed on identifying surface and membrane-associated proteins that are conserved across a range of rumen methanogens. The identification of potential vaccine antigens has been assisted by recent advances in the knowledge of rumen methanogen genomes. Methanogen surface proteins have been shown to be immunogenic in ruminants and vaccination of sheep with these proteins induced specific antibody responses in saliva and rumen contents. Current studies are directed towards identifying key candidate antigens and investigating the level and types of salivary antibodies produced in sheep and cattle vaccinated with methanogen proteins, stability of antibodies in the rumen and their impact on rumen microbial populations. In addition, there is a need to identify adjuvants that stimulate high levels of salivary antibody and are suitable for formulating with protein antigens to produce a low-cost and effective vaccine.

Rumen microbial (meta)genomics and its application to ruminant production.

Meat and milk produced by ruminants are important agricultural products and are major sources of protein for humans. Ruminant production is of considerable economic value and underpins food security in many regions of the world. However, the sector faces major challenges because of diminishing natural resources and ensuing increases in production costs, and also because of the increased awareness of the environmental impact of farming ruminants. The digestion of feed and the production of enteric methane are key functions that could be manipulated by having a thorough understanding of the rumen microbiome. Advances in DNA sequencing technologies and bioinformatics are transforming our understanding of complex microbial ecosystems, including the gastrointestinal tract of mammals. The application of these techniques to the rumen ecosystem has allowed the study of the microbial diversity under different dietary and production conditions. Furthermore, the sequencing of genomes from several cultured rumen bacterial and archaeal species is providing detailed information about their physiology. More recently, metagenomics, mainly aimed at understanding the enzymatic machinery involved in the degradation of plant structural polysaccharides, is starting to produce new insights by allowing access to the total community and sidestepping the limitations imposed by cultivation. These advances highlight the promise of these approaches for characterising the rumen microbial community structure and linking this with the functions of the rumen microbiota. Initial results using high-throughput culture-independent technologies have also shown that the rumen microbiome is far more complex and diverse than the human caecum. Therefore, cataloguing its genes will require a considerable sequencing and bioinformatic effort. Nevertheless, the construction of a rumen microbial gene catalogue through metagenomics and genomic sequencing of key populations is an attainable goal. A rumen microbial gene catalogue is necessary to understand the function of the microbiome and its interaction with the host animal and feeds, and it will provide a basis for integrative microbiome-host models and inform strategies promoting less-polluting, more robust and efficient ruminants.

Nitrogen metabolism and rumen microbial enumeration in lactating cows with divergent residual feed intake fed high-digestibility pasture.

Dairy cattle selected for negative residual feed intake (n-RFI; efficient) should maintain production while reducing dry matter intake over a lactation because of improvements in feed digestion and efficient use of nutrients. The objective of this study was to measure nitrogen (N) digestibility and rumen microbial community composition over a short period during early lactation in lactating Holstein-Friesian cows selected previously for divergent RFI. It was proposed that n-RFI cows would have greater apparent digestibility of N than the positive RFI (p-RFI; inefficient) animals, to compensate for the lower dry matter intake determined during selection for divergence. Sixteen 3-yr-old rumen-cannulated, lactating cows (56 ± 10d in milk) selected for n-RFI (n = 8) and p-RFI (n = 8) were housed in metabolism stalls and fed fresh vegetative ryegrass (Lolium perenne L.) pasture ad libitum as a sole diet during an 8-d digestibility study. Intake of nutrients and outputs of milk, feces, and urine were determined. Rumen parameters were determined by removing, weighing, and sampling digesta, and by cobalt-EDTA dilution. Intakes of N, dry matter, organic matter, or its components did not differ with RFI. Compared with p-RFI cows, n-RFI cows had a greater apparent N digestibility (77.2 vs. 75.5%), and a tendency toward greater dry matter and organic matter digestibilities. The n-RFI cows had a lower fecal N output (126 vs. 138 g/d) and a lower partition of feed N to fecal N (23.1 vs. 24.7%) compared with p-RFI animals. We found no differences between phenotypes in the partition of N to urinary N or milk crude protein but did observe a trend for n-RFI cows to partition less N to milk casein (16.8 vs. 17.9%). Rumen digesta mass was similar for both groups, despite differences in calculated fractional liquid outflow rates, and most bacterial, archaeal, protozoal, and fungal communities were similar for both phenotype groups. In conclusion, dry matter intake and rumen function were similar for both phenotypes when the animals were fed highly digestible fresh ryegrass, but apparent digestibility of dietary N was higher in the efficient (n-RFI) cows. Future research should measure digestion parameters in cows with divergent RFI when fed diets differing in chemical composition (e.g., divergent crude protein contents).

Development of a vaccine to mitigate greenhouse gas emissions in agriculture: vaccination of sheep with methanogen fractions induces antibodies that block methane production in vitro.

AIM: To develop an understanding of the immune responses of ruminants to methanogens, and to provide proof of a concept that harnessing the immune system of ruminants is a potentially viable approach to mitigate greenhouse gas emissions from agriculture. METHODS: Four subcellular fractions, namely cytoplasmic, two cell-wall preparations, and cell wall-derived proteins were prepared from Methanobrevibacter ruminantium M1. Twenty sheep (10 months of age) were vaccinated with these fractions or with whole cells (n=4 per group). Sheep were re-vaccinated once after 3 weeks, and antibody responses to M. ruminantium M1 antigens in sera and saliva measured using ELISA at 2 weeks after the second vaccination. Antigens recognised by the antisera were visualised using Western blotting. The antisera were tested in vitro for their impact on M. ruminantium M1, measuring the effect on cell growth, methane production, and ability to induce agglutination. RESULTS: Basal levels (pre-vaccination) of antibodies against M. ruminantium M1 antigens were low. Vaccination with the antigenic fractions induced strong antibody responses in serum. Both IgG and IgA responses to methanogen antigens were detected in saliva following vaccination. Western blot analysis of the antisera indicated reactivity of antibodies, and a wide range of proteins was present in the different methanogen fractions. Antisera against the various fractions agglutinated methanogens in an in-vitro assay. In addition, these antisera decreased the growth of a pure culture of a methanogen and production of methane in vitro. CONCLUSIONS: Antigens from methanogens are immunogenic in ruminants, and antisera from sheep vaccinated with fractions of methanogens have a significant impact on these organisms, inducing cell agglutination, and decreasing growth of methanogens and production of methane. Only antisera to selected methanogen fractions were able to achieve these effects. The results demonstrate the feasibility of a vaccination strategy to mitigate emission of methane.

A guidance for assessing and communicating uncertainties.

In the daily practice of science for policy, as experienced by governmental agencies which inform the policy and the public on the state and outlook of the environment, there is a pressing need for guidance in assessing and communicating uncertainties. This need extends beyond the quantitative assessment of uncertainties in model results, and focuses on the entire process of environmental assessment, running from problem framing towards reporting the results of the study. Using the Netherlands Environmental Assessment Agency (RIVM/MNP) as a case, the development, structure and content of such a guidance system is highlighted. Conditions for a successful implementation of the guidance system are discussed, and some prospects for future work are outlined.

Using the W&H integrated uncertainty analysis framework with non-initiated experts.

The W&H (Walker and Harremoës) integrated uncertainty analysis framework was put forward with the aim of providing a conceptual basis for the systematic treatment of uncertainty in model-based decision support activities such as policy analysis, integrated assessment and risk assessment. It provides a heuristic tool that can be applied in decision support exercises to classify and report the various dimensions of uncertainty. The intention is to stimulate better communication among analysts as well as between them and policymakers and stakeholders. The framework successfully articulates diverse scholarly understandings of 'uncertainty', 'ignorance', and 'quality' in science for policy, Nevertheless, experience with the W&H framework has revealed that many of the concepts put forward are relatively unfamiliar--and perhaps somewhat controversial--to experts practising decision support. Thus, efforts are required to communicate the W&H framework to experts in such a way that their knowledge of uncertainty is elicited adequately, without them being overly intimidated or confused by the novelty of the concepts presented to them. After introducing the W&H conceptual framework, this paper presents the methodology that was used in applying the W&H framework in expert elicitations on uncertainty in the risk assessment of genetically modified crops. Experiences with the use of this methodology are discussed and recommendations for further improvement are given.

Defluvicoccus vanus gen. nov., sp. nov., a novel Gram-negative coccus/coccobacillus in the 'Alphaproteobacteria' from activated sludge.

A novel Gram-negative coccus/coccobacillus, strain Ben 114(T), growing in tetrads, clusters or aggregates, was isolated from activated sludge by micromanipulation. 16S rRNA gene sequence analysis revealed that it belonged to the 'Alphaproteobacteria', with no close relatives among cultured bacterial isolates. On the basis of phylogenetic data, this organism is considered to belong to a new genus, Defluvicoccus, represented by the species Defluvicoccus vanus sp. nov., a name chosen because of the distinctive staining properties of this organism; only the cell wall stained strongly with a wide range of stains, giving the cell a hollow and empty appearance. No intracellular polyphosphate granules could be detected after staining, but poly-beta-hydroxyalkanoate inclusions were detected using Nile blue A staining. Because of its taxonomic distance from its closest relatives among the 'Alphaproteobacteria', namely members of the genera Azospirillum, Phaeospirillum, Rhodospirillum, Rhodocista, Magnetospirillum and Rhodospira, D. vanus is considered to represent a new phylogenetic lineage within subgroup 1 of the 'Alphaproteobacteria', the D. vanus subgroup. The type strain is Ben 114(T) (=NCIMB 13612(T)=CIP 107350(T)).

Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable.

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

Taxonomy of Australian clinical isolates of the genus Photorhabdus and proposal of Photorhabdus asymbiotica subsp. asymbiotica subsp. nov. and P. asymbiotica subsp. australis subsp. nov.

The relationship of Photorhabdus isolates that were cultured from human clinical specimens in Australia to Photorhabdus asymbiotica isolates from human clinical specimens in the USA and to species of the genus Photorhabdus that are associated symbiotically with entomopathogenic nematodes was evaluated. A polyphasic approach that involved DNA-DNA hybridization, phylogenetic analyses of 16S rRNA and gyrB gene sequences and phenotypic characterization was adopted. These investigations showed that gyrB gene sequence data correlated well with DNA-DNA hybridization and phenotypic data, but that 16S rRNA gene sequence data were not suitable for defining species within the genus Photorhabdus. Australian clinical isolates proved to be related most closely to clinical isolates from the USA, but the two groups were distinct. A novel subspecies, Photorhabdus asymbiotica subsp. australis subsp. nov. (type strain, 9802892T=CIP 108025T=ACM 5210T), is proposed, with the concomitant creation of Photorhabdus asymbiotica subsp. asymbiotica subsp. nov. Analysis of gyrB sequences, coupled with previously published data on DNA-DNA hybridization and PCR-RFLP analysis of the 16S rRNA gene, indicated that there are more than the three subspecies of Photorhabdus luminescens that have been described and confirmed the validity of the previously proposed subdivision of Photorhabdus temperata. Although a non-luminescent, symbiotic isolate clustered consistently with P. asymbiotica in gyrB phylogenetic analyses, DNA-DNA hybridization indicated that this isolate does not belong to the species P. asymbiotica and that there is a clear distinction between symbiotic and clinical species of Photorhabdus.

Severe Bordetella holmesii infection in a previously healthy adolescent confirmed by gene sequence analysis.

We describe an immunocompetent adolescent who presented with exceptionally severe Bordetella holmesii infection, including previously undescribed manifestations. Sequelae included a severe restrictive lung defect due to pulmonary fibrosis.

A member of the delta subgroup of proteobacteria from a pyogenic liver abscess is a typical sulfate reducer of the genus Desulfovibrio.

Strain FH26001/95 (ATCC 700045) was previously isolated from a pyogenic liver abscess from a human. Comparative 16S rRNA gene sequence analysis showed that this strain is related to members of the delta subgroup of the proteobacteria, within a cluster of sulfate-reducing bacteria (Desulfovibrio spp.) and non-sulfate-reducing bacteria (Bilophila wadsworthia and Lawsonia spp.). The phenotype of strain FH26001/95 was found to be typical of members of the genus Desulfovibrio. Growth and substrate transformations were possible at oxygen concentrations of 2 to 5% (vol/vol) but not at oxygen concentrations of 21% (vol/vol) in air. Its isolation from an infection in a human suggests that some members of the genus Desulfovibrio can be considered opportunistic pathogens.

Opitutus terrae gen. nov., sp. nov., to accommodate novel strains of the division 'Verrucomicrobia' isolated from rice paddy soil.

Three strains of obligately anaerobic bacteria were isolated from rice paddy soil microcosms. Comparative analysis of the 16S rRNA genes showed that these novel isolates have identical gene sequences and are members of the division 'Verrucomicrobia'. The novel strains are phenotypically and phylogenetically distinct from species described previously. One strain, PB90-1T, was characterized in more detail. The cells are cocci and are motile by means of a flagellum. Catalase and oxidase activities are absent. Growth-supporting substrates include mono-, di- and polysaccharides, while alcohols, amino acids and organic acids do not support growth. Propionate and acetate are the major end-products of fermentation. Nitrate is reduced to nitrite, but other external electron acceptors are not utilized. The G+C content of the genomic DNA is 74 mol%. This strain represents a taxon that has not yet been formally recognized, for which the name Opitutus terrae gen. nov., sp. nov. is proposed. The type strain is PB90-1T (= DSM 11246T).

Bacteremia due to Leptotrichia trevisanii sp. nov.

A thin, filamentous, non-motile, aerotolerant, anaerobic, gram-negative bacterium was isolated from the blood of a 46-year-old man who was diagnosed as having acute myeloid leukemia. The organism had a positive catalase reaction but was negative in indole and oxidase tests. A commercially available system failed to identify the bacterium, but 16S rRNA gene sequencing showed it to be most closely related (97% similarity) to a recently isolated Leptotrichia sp. The DNA base composition was 29.7% mol G+C, and the organism produced lactate as the sole end-product of glucose fermentation. These data indicate the isolate is a new species of Leptotrichia for which the name Leptotrichia trevisanii sp. nov. is proposed.