In vitro microbial inoculum: A review of its function and properties

This review considers microbial inocula used in in vitro systems from the perspective of their ability to degrade or ferment a particular substrate, rather than the microbial species that it contains. By necessity, this required an examination of bacterial, protozoal and fungal populations of the rumen and hindgut with respect to factors influencing their activity. The potential to manipulate these populations through diet or sampling time are examined, as is inoculum preparation and level. The main alternatives to fresh rumen fluid (i.e., caecal digesta or faeces) are discussed with respect to end-point degradabilities and fermentation dynamics. Although the potential to use rumen contents obtained from donor animals at slaughter offers possibilities, the requirement to store it and its subsequent loss of activity are limitations. Statistical modelling of data, although still requiring a deal of developmental work, may offer an alternative approach. Finally, with respect to the range of in vitro methodologies and equipment employed, it is suggested that a degree of uniformity could be obtained through generation of a set of guidelines relating to the host animal, sampling technique and inoculum preparation. It was considered unlikely that any particular system would be accepted as the 'standard' procedure. However, before any protocol can be adopted, additional data are required (e.g., a method to assess inoculum 'quality' with respect to its fermentative and/or degradative activity), preparation/inoculation techniques need to be refined and a methodology to store inocula without loss of efficacy developed. (c) 2005 Elsevier B.V. All rights reserved.

Risk factors for tick attachment to smallholder dairy cattle in Tanzania

A cross-sectional study was conducted in Tanga and Iringa regions of Tanzania, and a longitudinal study in Tanga, to investigate tick-control methods and other factors influencing tick attachment to the cattle of smallholder dairy farms. Most farmers reported applying acaricides at intervals of 1-2 weeks, most used acaricides that require on-farm dilution and most farmers incorrectly diluted the acaricides. Rhipicephalus appendiculatus and Boophilus spp. ticks were those most-frequently encountered on the cattle, but few cattle carried ticks of any species (only 13 and 4.6% of tick counts of the cattle yielded adult R. appendiculatus and Boophilus spp., respectively). Animals were more likely to carry one or more adult Boophilus spp. ticks if they also carried one or more R. appendiculatus adults (OR = 14.4, CI = 9.2, 22.5). The use of pour-on acaricides was associated with lower odds that animals carried a R. appendiculatus tick (OR = 0.29, CI = 0. 18, 0.49) but higher odds that they carried a Boophilus spp. tick (OR = 2.48, CI = 1.55, 3.97). Animals > 4 months old and those with a recent history of grazing had higher odds of carrying either a R. appendiculatus (ORs = 3.41 and 2.58, CIs = 2.34, 4.98 and 1.80, 3.71), or a Boophilus spp. tick (ORs = 5.70 and 2.18, CIs = 2.34, 4.98 and 1.49. 3.25), but zero-grazing management did not prevent ticks attaching to cattle even when combined with high-frequency acaricide treatments. The odds that animals carried ticks varied amongst the agro-ecological zones (AEZs) and administrative districts where the farms were situated-but there was still considerable residual variation in tick infestation at the farm level. (c) 2004 Elsevier B.V. All rights reserved.

Evolution of microbial virulence: the benefits of stress

Although genome sequencing of microbial pathogens has shed light on the evolution of virulence, the drivers of the gain and loss of genes and of pathogenicity islands (gene clusters), which contribute to the emergence of new disease outbreaks, are unclear. Recent experiments with the bean pathogen Pseudomonas syringae pv. phaseolicola illustrate how exposure to resistance mechanisms acts as the driving force for genome reorganization. Here we argue that the antimicrobial conditions generated by host defences can accelerate the generation of genome rearrangements that provide selective advantages to the invading microbe. Similar exposure to environmental stress outside the host could also drive the horizontal gene transfer that has led to the evolution of pathogenicity towards both animals and plants.

Development and application of in vivo expression technology (IVET) for analysing microbial gene expression in complex environments

Establishing the mechanisms by which microbes interact with their environment, including eukaryotic hosts, is a major challenge that is essential for the economic utilisation of microbes and their products. Techniques for determining global gene expression profiles of microbes, such as microarray analyses, are often hampered by methodological restraints, particularly the recovery of bacterial transcripts (RNA) from complex mixtures and rapid degradation of RNA. A pioneering technology that avoids this problem is In Vivo Expression Technology (IVET). IVET is a 'promoter-trapping' methodology that can be used to capture nearly all bacterial promoters (genes) upregulated during a microbe-environment interaction. IVET is especially useful because there is virtually no limit to the type of environment used (examples to date include soil, oomycete, a host plant or animal) to select for active microbial promoters. Furthermore, IVET provides a powerful method to identify genes that are often overlooked during genomic annotation, and has proven to be a flexible technology that can provide even more information than identification of gene expression profiles. A derivative of IVET, termed resolvase-IVET (RIVET), can be used to provide spatio-temporal information about environment-specific gene expression. More recently, niche-specific genes captured during an IVET screen have been exploited to identify the regulatory mechanisms controlling their expression. Overall, IVET and its various spin-offs have proven to be a valuable and robust set of tools for analysing microbial gene expression in complex environments and providing new targets for biotechnological development.

Comparison of the euryarchaeal microbial community in guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types

Termites are an important component of tropical soil communities and have a significant affect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physico-chemical conditions and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and Terminal-restriction Fragment Length Polymorphism (T-RFLP) analysis to examine methanogenic Archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If they are strictly vertically inherited, then MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus or Rice Cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities but these may represent transient populations in either guts or soil. Our data does not support the hypothesis that termite gut MA are derived from their food soil but also does not support a purely vertical transmission of gut microflora.

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an enterovirus of the Picornaviridae family, uses the complement regulator, decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF, consisting of short consensus repeat domains 3 and 4, from cryo-negative stain electron microscopy data (EMD #1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the two-fold symmetry axes. Despite this general similarity, our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB #1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF-binding phenotype in these viruses.

Dissociative electron attachment to dinitrogen pentoxide, N2O5

Electron attachment was studied in gaseous dinitrogen pentoxide, N2O5, for incident electron energies between a few meV and 10 eV. No stable parent anion N2O5- was observed but several anionic fragments (NO3-, NO2-, NO-, O-, and O-2(-)) were detected using quadrupole mass spectrometry. Many of these dissociative pathways were found to be coupled and provide detailed information on the dynamics of N2O5 fragmentation. Estimates of the cross sections for production of each of the anionic fragments were made and suggest that electron attachment to N2O5 is amongst the most efficient attachment reactions recorded for nonhalogenated polyatomic systems. (C) 2004 American Institute of Physics.

Place attachment and sense of belonging in the offices

Examines the concept of “place attachment” as defined in various disciplines and develops an effective conceptual approach that can be applied to facilities management. Describes the development of a model-matchmaking process adapted from Passini's model of cognitive mapping. Findings that the emergence of the new economy is undermining our ability to form attachments with people, places and companies. However, one of the unintended effects of this is that it has strengthened the value of place and aroused a longing for community. Moreover, loyalty to an organisation is increasingly determined by social and place attachment. Proposes that further research needs to be undertaken to “engineer out” the negative impacts of flexibility associated with loss of place. States that place attachment presents a challenging view of the world that is contrary to all the received wisdom in facilities management, where flexibility has always assumed an unchallenged position in relation to buildings and people. Concludes that this research area presents many pragmatic design and operational questions for facilities managers.

Microbial species involved in production of 1,2-sn-diacylglycerol and effects of phosphatidylcholine on human fecal microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKQ, which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.

Post-genomics approaches towards monitoring changes within the microbial ecology of the gut

The human gut microbiota, comprising many hundreds of different microbial species, has closely co-evolved with its human host over the millennia. Diet has been a major driver of this co-evolution, in particular dietary non-digestible carbohydrates. This dietary fraction reaches the colon and becomes available for microbial fermentation, and it is in the colon that the great diversity of gut microorganisms resides. For the vast majority of our evolutionary history humans followed hunter-gatherer life-styles and consumed diets with many times more non-digestible carbohydrates, fiber and whole plant polyphenol rich foods than typical Western style diets today.

Relationship between sublethal injury and microbial inactivation by the combination of high hydrostatic pressure and citral or tert-butyl hydroquinone

The aim was to investigate (i) the occurrence of sublethal injury in Listeria monocytogenes, Escherichia coli, and Saccharomyces cerevisiae after high hydrostatic pressure (HHP) treatment as a function of the treatment medium pH and composition and (ii) the relationship between the occurrence of sublethal injury and the inactivating effect of a combination of HHP and two antimicrobial compounds, tert-butyl hydroquinone (TBHQ) and citral. The three microorganisms showed a high proportion of sublethally injured cells (up to 99.99% of the surviving population) after HHP. In E. coli and L. monocytogenes, the extent of inactivation and sublethal injury depended on the pH and the composition of the treatment medium, whereas in S. cerevisiae, inactivation and sublethal injury were independent of medium pH or composition under the conditions tested. TBHQ alone was not lethal to E. coli or L. monocytogenes but acted synergistically with HHP and 24-h refrigeration, resulting in a viability decrease of >5 log(10) cycles of both organisms. The antimicrobial effect of citral depended on the microorganism and the treatment medium pH. Acting alone for 24 h under refrigeration, 1,000 ppm of citral caused a reduction of 5 log(10) cycles of E. coli at pH 7.0 and almost 3 log(10) cycles of L. monocytogenes at pH 4.0. The combination of citral and HHP also showed a synergistic effect. Our results have confirmed that the detection of sublethal injury after HHP may contribute to the identification of those treatment conditions under which HHP may act synergistically with other preserving processes.

Effect of dietary phytate and microbial phytase on mineral and trace element bioavailability- a literature review

Phytic acid (PA) is the main phosphorus storage compound in cereals, legumes and oil seeds. In human populations where phytate-rich cereals such as wheat, maize and rice are a staple food, phytate may lead to mineral and trace element deficiency. Zinc appears to be the trace element whose bioavailability is most influenced by PA. Furthermore, several studies in humans as well as in monogastric animals clearly indicate an inhibition of non-haem iron absorption at marginal iron supply due to phytic acid. In fact PA seems to be, at least partly, responsible for the low absorption efficiency and high incidence of iron deficiency anaemia evident in most developing countries, where largely vegetarian diets are consumed Microbial phytases have provided a realistic means of improving mineral availability from traditionally high-phytate diets. In fact it has been consistently shown that Aspergillus phytases significantly enhance the absorption of calcium, magnesium and zinc in pigs and rats. Furthermore there are a few studies in humans indicating an improvement of iron bioavailability due to microbial phytase.