Toxicity of mono-, di- and tri-chlorophenols to lux marked terrestrial bacteria, Burkholderia species Rasc c2 and Pseudomonas fluorescens

Burkholderia species RASC and Pseudomonas fluorescens were marked with lux genes, encoding for bioluminescence and used to assess the toxicity of mono-, di- and tri-chlorophenols by determining the decline in bioluminescence following exposure to the compounds in aqueous solution. Toxicity was expressed as a 50% effective concentration value (EC50, equating to the concentration of compound which caused a 50% decline in bioluminescence. Comparing the toxicity values of the compounds showed that, in general, increasing the degree of chlorination, increased toxicity. By carrying out forward multiple linear regressions with log10 EC50 values and physio-chemical descriptors, it was shown that molecular parameters describing the hydrogen bonding nature of a chlorophenol provided a better fit than regressions between toxicity data and log10 Kow alone. Utilising these descriptor variables in equations, it was shown that the toxicity of chlorophenols to the lux marked bacteria could be predicted from the compounds physio-chemical characteristics. By correlating lux marked RASC c2 and P. fluorescens EC50 values with toxicity values using Pimephales promelas (fathead minnow), Tetrahymena pyriformis (ciliate) and marine bacterium Vibriofischeri, it was apparent that lux marked RASC c2 correlated well with the freshwater aquatic species (P. promelas and T. pyriformis). This implied that for predictions of toxicity of organic xenobiotic compounds to higher organisms, lux marked RASC c2 could be utilised as a rapid surrogate.

Quantitative structure-toxicity relationships for chlorophenols to bioluminescent lux-marked bacteria using atom-based semi-empirical molecular-orbital descriptors

Literature data on the toxicity of chlorophenols for three luminescent bacteria (Vibrio fischeri, and the lux-marked Pseudomonas fluorescens 10586s pUCD607 and Burkholderia spp. RASC c2 (Tn4431)) have been analyzed in relation to a set of computed molecular physico-chemical properties. The quantitative structure-toxicity relationships of the compounds in each species showed marked differences when based upon semi-empirical molecular-orbital molecular and atom based properties. For mono-, di- and tri-chlorophenols multiple linear regression analysis of V. fischeri toxicity showed a good correlation with the solvent accessible surface area and the charge on the oxygen atom. This correlation successfully predicted the toxicity of the heavily chlorinated phenols, suggesting in V. fischeri only one overall mechanism is present for all chlorophenols. Good correlations were also found for RASC c2 with molecular properties, such as the surface area and the nucleophilic super-delocalizability of the oxygen. In contrast the best QSTR for P. fluorescens contained the 2nd order connectivity index and ELUMO suggesting a different, more reactive mechanism. Cross-species correlations were examined, and between V. fischeri and RASC c2 the inclusion of the minimum value of the nucleophilic susceptibility on the ring carbons produced good results. Poorer correlations were found with P. fluorescens highlighting the relative similarity of V. fischeri and RASC c2, in contrast to that of P. fluorescens.

Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation

Background: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC).

Methodology/Principal Findings: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF Delta ARE and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells.

Conclusions/Significance: We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.

Inflammatory bowel disease, gut bacteria and probiotic therapy

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD) and both diseases lead to high morbidity and health care costs. Complex interactions between the immune system, enteric commensal bacteria and host genotype are thought to underlie the development of IBD although the precise aetiology of this group of diseases is still unknown. The understanding of the composition and complexity of the normal gut microbiota has been greatly aided by the use of molecular methods and is likely to be further increased with the advent of metagenomics and metatranscriptomics approaches, which will allow an increasingly more holistic assessment of the microbiome with respect to both diversity and function of the commensal gut microbiota. Studies thus far have shown that the intestinal microbiota drives the development of the gut immune system and can induce immune homeostasis as well as contribute to the development of IBD. Probiotics which deliver some of the beneficial immunomodulatory effects of the commensal gut microbiota and induce immune homeostasis have been proposed as a suitable treatment for mild to moderate IBD. This review provides an overview over the current understanding of the commensal gut microbiota, its interactions with the mucosal immune system and its capacity to induce both gut homeostasis as well as dysregulation of the immune system. Bacterial-host events, including interactions with pattern recognition receptors (PRRs) expressed on epithelial cells and dendritic cells (DCs) and the resultant impact on immune responses at mucosal surfaces will be discussed. (C) 2009 Elsevier GmbH. All rights reserved.

Therapeutic Effects of Human Mesenchymal Stem Cells in Ex Vivo Human Lungs Injured with Live Bacteria

Rationale: Mesenchymal stem cells secrete paracrine factors that can regulate lung permeability and decrease inflammation, making it a potentially attractive therapy for acute lung injury. However, concerns exist whether mesenchymal stem cells' immunomodulatory properties may have detrimental effects if targeted toward infectious causes of lung injury. Objectives: Therefore, we tested the effect of mesenchymal stem cells on lung fluid balance, acute inflammation, and bacterial clearance. Methods: We developed an Escherichia coli pneumonia model in our ex vivo perfused human lung to test the therapeutic effects of mesenchymal stem cells on bacterial-induced acute lung injury. Measurements and Main Results: Clinical-grade human mesenchymal stem cells restored alveolar fluid clearance to a normal level, decreased inflammation, and were associated with increased bacterial killing and reduced bacteremia, in part through increased alveolar macrophage phagocytosis and secretion of antimicrobial factors. Keratinocyte growth factor, a soluble factor secreted by mesenchymal stem cells, duplicated most of the antimicrobial effects. In subsequent in vitro studies, we discovered that human monocytes expressed the keratinocyte growth factor receptor, and that keratinocyte growth factor decreased apoptosis of human monocytes through AKT phosphorylation, an effect that increased bacterial clearance. Inhibition of keratinocyte growth factor by a neutralizing antibody reduced the antimicrobial effects of mesenchymal stem cells in the ex vivo perfused human lung and monocytes grown in vitro injured with E. coli bacteria. Conclusions: In E. coli-injured human lungs, mesenchymal stem cells restored alveolar fluid clearance, reduced inflammation, and exerted antimicrobial activity, in part through keratinocyte growth factor secretion.

Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.

Biomolecular mechanisms of staphylococcal biofilm formation

The multitude of biomolecular and regulatory factors involved in staphylococcal adhesion and biofilm formation owe much to their ability to colonize surfaces, allowing the biofilm form to become the preferential bacterial phenotype. Judging by total number, biomass and variety of environments colonized, bacteria can be categorized as the most successful lifeform on earth. This is due to the ability of bacteria and other microorganisms to respond phenotypically via biomolecular processes to the stresses of their surrounding environment. This review focuses on the specific pathways involved in the adhesion of the Gram-positive bacteria Staphylococcus epidermidis and Staphylococcus aureus with reference to the role of specific cell surface adhesins, the ica operon, accumulation-associated proteins and quorum-sensing systems and their significance in medical device-related infection.

A preliminary survey of micro-organisms in the gut and pellets of a tropical millipede Doratogonus uncinatus Attems (Diplopoda, Spirostreptida, Spirostreptidae).

Millipede gut microbiology and decomposition of faecal pellets over a period of eight weeks were studied in the laboratory. Bacterial numbers, carbon and nitrogen content, pH and weight loss were monitored. Heterotrophic bacteria were the most abundant and reached a peak in the first two weeks of decomposition. The amount of carbon was constant while ammonium nitrogen decreased from 1.51 % to 0.03 % after eight weeksThe pH of the pellets was slightly acidic and did not change much during the course of decomposition. A succession of micro-organisms was observed on decomposing pellets. Zygomycetes were replaced by Ascomycetes after 20 days of decomposition. Decomposition was significantly affected by temperature. The rate of decomposition was highest at 35[degree]C .

Dual Enlargement of Gold Nanoparticles: From Mechanism to Scanometric Detection of Pathogenic Bacteria

A mechanism of dual enlargement of gold nanoparticles (AuNPs) comprising two steps is described. In the first step, the AuNPs are enlarged by depositing Au atoms on their crystalline faces. In this process, the particles are not only enlarged but they are also observed to multiply: new Au nuclei are formed by the budding and division of the enlarged particles. In the second step, a silver enhancement is subsequently performed by the deposition of silver atoms on the enlarged and newly formed AuNPs to generate bimetallic Au@Ag core-shell structures. The dual nanocatalysis greatly enhances the electron density of the nanostructures, leading to a stronger intensity for colorimetric discrimination as well as better sensitivity for quantitative measurement. Based on this, a simple scanometric assay for the on-slide detection of the food-born pathogen Campylobacter jejuni is developed. After capturing the target bacteria, gold-tagged immunoprobes are added to create a signal on a solid substrate. The signal is then amplified by the dual enlargement process, resulting in a strong color intensity that can easily be recognized by the unaided eye, or measured by an inexpensive flatbed scanner. In this paper, dual nanocatalysis is reported for the first time. It provides a valuable mechanistic insight into the development of a simple and cost-effective detection format.

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

Tracking the Fate of Microbially Sequestered Carbon Dioxide in Soil Organic Matter

The microbial contribution to soil organic matter (SOM) has recently been shown to be much larger than previously thought and thus its role in carbon sequestration may also be underestimated. In this study we employ C-13 ((CO2)-C-13) to assess the potential CO2 sequestration capacity of soil chemoautotrophic bacteria and combine nuclear magnetic resonance (NMR) with stable isotope probing (SIP), techniques that independently make use of the isotopic enrichment of soil microbial biomass. In this way molecular information generated from NMR is linked with identification of microbes responsible for carbon capture. A mathematical model is developed to determine real-time CO2 flux so that net sequestration can be calculated. Twenty-eight groups of bacteria showing close homologies with existing species were identified. Surprisingly, Ralstonia eutropha was the dominant group. Through NMR we observed the formation of lipids, carbohydrates, and proteins produced directly from CO2 utilized by microbial biomass. The component of SOM directly associated with CO2 capture was calculated at 2.86 mg C (89.21 mg kg(-1)) after 48 h. This approach can,differentiate between SOM derived through microbial uptake of CO2 and other SOM constituents and represents a first step in tracking the fate and dynamics of microbial biomass in soil.