Diarrhea-associated biofilm formed by enteroaggregative <Emphasis Type="Italic">Escherichia coli</Emphasis> and aggregative <Emphasis Type="Italic">Citrobacter freundii</Emphasis>: a consortium mediated by putative F pili

Background  

Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea.     

New Device for High-Throughput Viability Screening of Flow Biofilms

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.Bacterial biofilms are surface-attached communities that are encased in a polymeric matrix, which exhibit a high degree of resistance to antimicrobial agents and the host immune system (12, 16). This makes them medically important; diseases with a biofilm component are chronic and difficult to eradicate. Examples of such diseases are cystitis (1), endocarditis (31), cystic fibrosis (35), and middle-ear (17) and indwelling medical device-associated (20) infections. Biofilms also play important environmental roles in, for example, wastewater treatment (38), bioremediation (29, 30), biofouling (7), and biocorrosion (2). Better control of biofilms requires elucidation of the molecular basis of their superior resistance (by identifying resistance-compromised mutants) and identification of compounds with antibiofilm activity. While our understanding of these aspects of biofilms has increased (11, 15, 25-27, 36), further work, including development of accurate high-throughput (HTP) methods for screening biofilm viability, is needed.Two major biofilm models are studied in the laboratory, biofilms grown without a continuous flow of fresh medium and biofilms grown with a continuous flow of fresh medium; examples of these two models are microtiter well biofilms and flow cell biofilms, respectively. Methods have been developed for HTP screening of the viability of static biofilms (6, 28, 32, 33), but there are no methods for HTP screening of flow biofilms. The latter biofilms are typically grown in flow cells, which have to be examined individually to determine viability and thus cannot be used for rapid screening. An HTP screening method for flow biofilms is desirable, as these biofilms more closely approximate natural biofilms and can differ from static biofilms evidently due to hydrodynamic influences on cell signaling (22, 34). For example, the ability of rpoS-deficient Escherichia coli (lacking σ^S) to form flow biofilms is impaired, but its capacity to form biofilms under static conditions is enhanced (18).We describe here a new application of a recently developed device (8-10, 13), the “BioFlux” device consisting of microfluidic channels for biofilm growth. Other microfluidic devices have recently been used for biofilm formation (14, 19, 21, 23), but none of them has been used for HTP screening. The BioFlux device permits rapid measurement of the fluorescence of flow biofilms with a plate reader, which permits initial HTP screening of the viability of such biofilms.     

Transcriptional robustness and protein interactions are associated in yeast

Background

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease with a varying range of phenotypes involving abnormal vasculature primarily manifested as arteriovenous malformations in various organs, including the nose, brain, liver, and lungs. The varied presentation and involvement of different organ systems makes the choice of potential treatment medications difficult.

Results

A patient with a mixed-clinical presentation and presumed diagnosis of HHT, severe exertional dyspnea, and diffuse pulmonary shunting at the microscopic level presented for treatment. We sought to analyze her metabolomic plasma profile to assist with pharmacologic treatment selection. Fasting serum samples from 5 individuals (4 healthy and 1 with HHT) were metabolomically profiled. A global metabolic network reconstruction, Recon 1, was used to help guide the choice of medication via analysis of the differential metabolism between the patient and healthy controls using metabolomic data. Flux Balance Analysis highlighted changes in metabolic pathway activity, notably in nitric oxide synthase (NOS), which suggested a potential link between changes in vascular endothelial function and metabolism. This finding supported the use of an already approved medication, bevacizumab (Avastin). Following 2 months of treatment, the patient's metabolic profile shifted, becoming more similar to the control subject profiles, suggesting that the treatment was addressing at least part of the pathophysiological state.

Conclusions

In this 'individualized case study' of personalized medicine, we carry out untargeted metabolomic profiling of a patient and healthy controls. Rather than filtering the data down to a single value, these data are analyzed in the context of a network model of metabolism, in order to simulate the biochemical phenotypic differences between healthy and disease states; the results then guide the therapy. This presents one approach to achieving the goals of individualized medicine through Systems Biology and causal models analysis.     

Regionally differentiated estimates of cropland N2O emissions reduce uncertainty in global calculations

Nitrogen (N) additions to cropland soils are the largest source of anthropogenic nitrous oxide (N₂O) emissions and are an important contributor to global greenhouse gas radiative forcing. Progress in understanding controls on N₂O fluxes from soils is demonstrated in increasingly sophisticated emissions estimates with improved spatial and source resolution. These methods build upon ongoing field, laboratory, and modeling advances that are restricted to just a handful of countries. Thus, burgeoning new knowledge is of limited utility for improving N₂O emissions estimates for the rest of the world where prospects for near‐term advances are constrained by the limited breadth of observations and availability of model driver data. Here, we use Bayesian inversion to leverage information from recent national‐level N₂O emission inventories and reduce uncertainty by up to 65% for estimates of regional and global direct cropland N₂O emissions. Our estimates for the proportion of N inputs lost as N₂O vary by a factor of two between regions and depart from existing default emission factors, yet regional emissions estimates based on these factors are consistent with global, regional, and local observations. Improved regional emission factors will enhance national greenhouse gas inventories in information‐poor countries and guide efforts to reduce agricultural N₂O emissions.     

A Chlamydia pneumoniae-specific peptide induces experimental autoimmune encephalomyelitis in rats

It has been reported recently that the bacterial respiratory pathogen Chlamydia pneumoniae is present in the cerebrospinal fluid of a subset of multiple sclerosis (MS) patients. However, it is not known whether this organism is a causative agent of MS, or merely an opportunistic pathogen that takes advantage of a disease process initiated by some other means. We report identification of a 20-mer peptide from a protein specific to C. pneumoniae which shares a 7-aa motif with a critical epitope of myelin basic protein, a major CNS Ag targeted by the autoimmune response in MS. This bacterial peptide induces a Th1 response accompanied by severe clinical and histological experimental autoimmune encephalomyelitis in Lewis rats, a condition closely reflective of many aspects of MS. Studies with peptide analogues suggest that different populations of encephalitogenic T cells are activated by the C. pneumoniae and myelin basic protein Ags. Mild experimental autoimmune encephalomyelitis was also observed when rats were immunized with sonicated C. pneumoniae in CFA.     

The innate immune repertoire in Cnidaria - ancestral complexity and stochastic gene loss

Background  

Characterization of the innate immune repertoire of extant cnidarians is of both fundamental and applied interest - it not only provides insights into the basic immunological 'tool kit' of the common ancestor of all animals, but is also likely to be important in understanding the global decline of coral reefs that is presently occurring. Recently, whole genome sequences became available for two cnidarians, Hydra magnipapillata and Nematostella vectensis, and large expressed sequence tag (EST) datasets are available for these and for the coral Acropora millepora.