Modulation of chemokine gene expression by Shiga-toxin producing Escherichia coli belonging to various origins and serotypes

Infection with Shiga-toxin producing Escherichia coli (STEC) may result in the development of the haemolytic-uremic syndrome (HUS), the main cause of acute renal failure in children. While O157:H7 STEC are associated with large outbreaks of HUS, it is difficult to predict whether a non-O157:H7 isolate can be pathogenic for humans. The mucosal innate immune response plays a central role in the pathogenesis of HUS; therefore, we compared the induction of IL-8 and CCL20 in human colon epithelial cells infected with strains belonging to different serotypes, isolated from cattle or from HUS patients. No correlation was observed between strain virulence and chemokine gene expression. Rather, the genetic background of the strains seems to determine the chemokine gene expression profile. Investigating the contribution of different bacterial factors in this process, we show that the type III secretion system of O157:H7 bacteria, but not the intimate adhesion, is required to stimulate the cells. In addition, H7, H10, and H21 flagellins are potent inducers of chemokine gene expression when synthesized in large amount.

On-chip non-invasive and label-free cell discrimination by impedance spectroscopy

OBJECTIVES: Many flow-cytometric cell characterization methods require costly markers and colour reagents. We present here a novel device for cell discrimination based on impedance measurement of electrical cell properties in a microfluidic chip, without the need of extensive sample preparation steps and the requirement of labelling dyes. MATERIALS AND METHODS, RESULTS: We demonstrate that in-flow single cell measurements in our microchip allow for discrimination of various cell line types, such as undifferentiated mouse fibroblasts 3T3-L1 and adipocytes on the one hand, or human monocytes and in vitro differentiated dendritic cells and macrophages on the other hand. In addition, viability and apoptosis analyses were carried out successfully for Jurkat cell models. Studies on several species, including bacteria or fungi, demonstrate not only the capability to enumerate these cells, but also show that even other microbiological life cycle phases can be visualized. CONCLUSIONS: These results underline the potential of impedance spectroscopy flow cytometry as a valuable complement to other known cytometers and cell detection systems.

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-gamma-primed eosinophils to release mitochondrial DNA in a reactive oxygen species-dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner--in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.

Isolation of plant growth promoting bacteria from torch lake sediments and their role in phytoremediation of metal contaminated soil

In this study, we isolated eight copper-resistant bacteria from Torch Lake sediment contaminated by copper mine tailings (stamp sand). Sequence analysis of gyrB and rpoD genes revealed that these organisms are closer to various Pseudomonas species. These eight bacterial isolates were also resistant to zinc, cesium, lead, arsenate and mercury. Further characterization showed that all the strains produced plant growth promoting indole-3-acetic acid (IAA), iron chelating siderophore and solubilized mineral phosphate and metals. The effect of bacterial inoculation on plant growth and copper uptake by maize (Zea mays) and sunflower (Helianthus annuus) was investigated using one of the isolates (Pseudomonas sp. TLC 6-6.5-4) with higher IAA production and phosphate and metal soubilization, which resulted in a significant increase in copper accumulation in maize and sunflower, and an increase in the total biomass of maize. Genes involved in copper resistance of Pseudomonas sp. TLC 6-6.5-4 was analyzed by transposon mutational analysis. Two copper sensitive mutants with significant reduction in copper resistance were identified: CSM1, a mutant disrupted in trp A gene (tryptophan synthase alpha subunit); CSM2, a mutant disrupted in clpA gene (ATP-dependent Clp protease). Proteomic and metabolomic analysis were performed to identify biochemical and molecular mechanisms involved in copper resistance using CSM2 due to its lower minimum inhibitory concentration compared with CSM1 and the wild type. The effect of different bacterial inoculation methods on plant growth, copper uptake and soil enzyme activities was investigated. Four different delivery methods were used including soil inoculation (before or after plant emergence), seed coating and root dipping. Soil inoculation before sowing seeds and coating seeds with PGPB led to better growth of maize, higher copper uptake and an increase in soil invertase and dehydrogenase activities. Proteomic and metabolomic analyses were performed to investigate the effect of bacterial inoculation on maize grown in normal soil and stamp sand. Our results revealed that bacterial inoculation led to environment-dependent effects on maize proteome and metabolome.

REMEDIATION OF TETRACYCLINE FROM WATER SOURCES USING VETIVER GRASS (Chrysopogon zizanioides L. Nash) AND TETRACYCLINE-TOLERANT ROOT-ASSOCIATED BACTERIA

Antibiotics are emerging contaminants worldwide. Due to insufficient policy regulations, public awareness, and the constant exposure of the environment to antibiotic sources has created a major environmental concern. Wastewater treatment plants (WWTP) are not equipped to filter-out these compounds before the discharge of the disinfected effluent into water sources (e.g., lakes and streams) and current available technologies are not equipped to remediate these compounds from environmental sources. Hence, the challenge remains to establish a biological system to remove these antibiotics from wastewater. An invitro hydroponic remediation system was developed using vetiver grass (Chrysopogon zizanioides L. Nash) to remediate tetracycline (TC) from water. Comparative metabolomics studies were conducted to investigate the metabolites/pathways associated with tetracycline metabolism in plants and TC-degrading bacteria. The results show that vetiver plants effectively uptake tetracycline from water sources. Vetiver root-associated bacteria recovered during the hydroponic remediation trial were highly tolerant to TC (as high as 600 ppm) and could use TC as a sole carbon and energy source. Growth conditions (pH, temperature, and oxygen requirement) for TC-tolerant bacteria were optimized for higher TC remediation capability from water sources. The plant (roots and shoots) and bacterial species were further characterized for the metabolites produced during the TC degradation process using GC-MS to identify the possible biochemical mechanism involved. Also, the plant root zone was screened for metabolites/enzymes that were secreted during antibiotic degradation and could potentially enhance the degradation process. The root zone was selected for this analysis because this region of the plant has shown a greater capacity for antibiotic degradation compared to the shoot zone. The role of antioxidant enzymes in TC degradation process revealed glutathione-S-transferase (GSTs) as an important group of enzymes in both plant and bacteria potentially involved in TC degradation process. Metabolomics results also suggest potential GST activity in the TC remediation/ transformation process used by plants. This information could be useful in gaining insights for the application of biological remediation systems for the mitigation of antibiotics from waste-water.

Incorporation of 2,4,6-trinitrotoluene (TNT) transforming bacteria into explosive formulations

Pseudomonas putida GG04 and Bacillus SF have been successfully incorporated into an explosive formulation to enhance biotransformation of TNT residues and/or explosives which fail to detonate due to technical faults. The incorporation of the microorganisms into the explosive did not affect the quality of the explosive (5 years storage) in terms of detonation velocity while complete biotransformation of TNT moieties upon transfer in liquid media was observed after 5 days. The incorporated microorganisms reduced TNT sequentially leading to the formation of hydroxylaminodinitrotoluenes (HADNT), 4-amino-2,6-dinitrotoluenes; 2-amino-4,6-dinitrotoluenes, different azoxy compounds; 2,6-diaminonitrotoluenes (2,4-DAMNT) and 2,4-diaminonitrotoluenes (2,6-DAMNT). However, the accumulation of AMDNT and DAMNT (major dead-end metabolites) was effectively prevented by incorporating guaiacol and catechol during the biotransformation process.

Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I

DNA ligases are important enzymes which catalyze the joining of nicks between adjacent bases of double-stranded DNA. NAD1-dependent DNA ligases (LigA) are essential in bacteria and are absent in humans. They have therefore been identified as novel, validated and attractive drug targets. Using virtual screening against an in-house database of compounds and our recently determined crystal structure of the NAD1 binding domain of the Mycobacterium tuberculosis LigA, we have identified N1, Nn-bis-(5-deoxy-a-D-xylofuranosylated) diamines as a novel class of inhibitors for this enzyme. Assays involving M.tuberculosis LigA, T4 ligase and human DNA ligase I show that these compounds specifically inhibit LigA from M.tuberculosis. In vitro kinetic and inhibition assays demonstrate that the compounds compete with NAD1 for binding and inhibit enzyme activity with IC50 values in the mM range. Docking studies rationalize the observed specificities and show that among several glycofuranosylated diamines, bis xylofuranosylated diamines with aminoalkyl and 1, 3-phenylene carbamoyl spacers mimic the binding modes of NAD1 with the enzyme. Assays involving LigA-deficient bacterial strains show that in vivo inhibition of ligase by the compounds causes the observed antibacterial activities. They also demonstrate that the compounds exhibit in vivo specificity for LigA over ATPdependent ligase. This class of inhibitors holds out the promise of rational development of new anti-tubercular agents.

Identification of animal Pasteurellaceae by MALDI-TOF mass spectrometry

Species of the family Pasteurellaceae play an important role as primary or opportunistic animal pathogens. In veterinary diagnostic laboratories identification of this group of bacteria is mainly done by phenotypic assays while genetic identification based on housekeeping genes is mostly used for research and particularly important diagnostic samples. MALDI-TOF MS seems to represent a promising alternative to the currently practiced cumbersome, phenotypic diagnostics carried out in many veterinary diagnostic laboratories. We therefore assessed its application for animal associated members of the family Pasteurellaceae. The Bruker Biotyper 3.0 database was complemented with reference spectra of clinically relevant as well as commensal animal Pasteurellaceae species using generally five strains per species or subspecies and tested for its diagnostic potential with additional, well characterized field isolates. About 250 strains comprising 15 genera and more than 40 species and subspecies were included in the study, covering most representatives of the family. A high discrimination at the genus and species level was observed. Problematic discrimination was only observed with some closely related species and subspecies. MALDI-TOF MS was shown to represent a highly potent method for the diagnosis of this group of animal pathogens, combining speed, precision and low running costs.

Routine phenotypic identification of bacterial species of the family Pasteurellaceae isolated from animals

Pasteurellaceae are bacteria with an important role as primary or opportunistic, mainly respiratory, pathogens in domestic and wild animals. Some species of Pasteurellaceae cause severe diseases with high economic losses in commercial animal husbandry and are of great diagnostic concern. Because of new data on the phylogeny of Pasteurellaceae, their taxonomy has recently been revised profoundly, thus requiring an improved phenotypic differentiation procedure to identify the individual species of this family. A new and simplified procedure to identify species of Actinobacillus, Avibacterium, Gallibacterium, Haemophilus, Mannheimia, Nicoletella, and Pasteurella, which are most commonly isolated from clinical samples of diseased animals in veterinary diagnostic laboratories, is presented in the current study. The identification procedure was evaluated with 40 type and reference strains and with 267 strains from routine diagnostic analysis of various animal species, including 28 different bacterial species. Type, reference, and field strains were analyzed by 16S ribosomal RNA (rrs) and rpoB gene sequencing for unambiguous species determination as a basis to evaluate the phenotypic differentiation schema. Primary phenotypic differentiation is based on beta-nicotinamide adenine dinucleotide (beta-NAD) dependence and hemolysis, which are readily determined on the isolation medium. The procedure divides the 28 species into 4 groups for which particular biochemical reactions were chosen to identify the bacterial species. The phenotypic identification procedure allowed researchers to determine the species of 240 out of 267 field strains. The procedure is an easy and cost-effective system for the rapid identification of species of the Pasteurellaceae family isolated from clinical specimens of animals.

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Estrogen Modulates Hepatic Gene Expression and Survival of Rainbow Trout Infected with Pathogenic Bacteria Yersinia ruckeri

In the aquatic environment, fish are exposed to various stimuli at once and have developed different response mechanisms to deal with these multiple stimuli. The current study assessed the combined impacts of estrogens and bacterial infection on the physiological status of fish. Juvenile rainbow trout were exposed to two different concentrations of 17 beta-estradiol (E2) (2 or 20 mg/kg feed) and then infected with three concentrations of Yersinia ruckeri, a bacterial pathogen causing massive losses in wild and farmed salmonid populations. Organism-level endpoints to assess the impact of the single and combined treatments included hepatic vitellogenin transcript expression to evaluate the E2 exposure efficiency and survival rate of pathogen-challenged fish. The two E2 doses increased vitellogenin levels within the physiological range. Infection with Y. ruckeri caused mortality of trout, and this effect was significantly enhanced by a simultaneous exposure to high E2 dose. The hormone reduced survival at intermediate and high (10(4) and 10(6) colony forming units, cfu) bacterial concentrations, but not for a low one (10(2) cfu). Analysis of hepatic gene expression profiles by a salmonid 2 k cDNA microarray chip revealed complex regulations of pathways involved in immune responses, stress responses, and detoxicification pathways. E2 markedly reduced the expression of several genes implicated in xenobiotic metabolism. The results suggest that the interaction between pathogen and E2 interfered with the fish's capability of clearing toxic compounds. The findings of the current study add to our understanding of multiple exposure responses in fish.

Antibacterial activity of moxifloxacin on bacteria associated with periodontitis within a biofilm

The activity of moxifloxacin was compared with ofloxacin and doxycycline against bacteria associated with periodontitis within a biofilm (single strain and mixed population) in vitro. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of moxifloxacin, ofloxacin and doxycyline were determined against single strains and mixed populations in a planktonic state. Single-species biofilms of two Porphyromonas gingivalis and two Aggregatibacter actinomycetemcomitans strains and a multi-species biofilm consisting of 12 species were formed for 3 days. The minimal biofilm eradication concentrations (MBECs) were determined after exposing the biofilms to the antibacterials (0.002 - 512 µg ml-1) for 18 h, addition of nutrient broth for 3 days and subsequent subcultivation. Photographs were taken by using confocal laser scanning microscopy and scanning electron microscopy. The MICs and MBCs did not differ between ofloxacin and moxifloxacin against A. actinomycetemcomitans, moxifloxacin was more active than the other tested antibacterials against anaerobes and the mixed population. The single-species biofilms were eradicated by moderate concentrations of the antibacterials, the lowest MBECs were always found for moxifloxacin (2-8 µg ml-1). MBECs against the multi-species biofilms were 128 µg ml-1, >512 µg ml-1 and >512 µg ml-1 for moxifloxacin, ofloxacin and doxycycline, respectively. In summary, moxifloxacin in a topical formulation may have potential as an adjunct to mechanical removal of the biofilms.

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.

Congenital thrombotic thrombocytopenic purpura caused by new compound heterozygous mutations of the ADAMTS13 gene

Upshaw-Schulman syndrome (USS) is due to severe congenital deficiency of von Willebrand factor (VWF)-cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 domains, nr 13) activity resulting in the presence of unusually large forms of VWF in the circulation, causing intravascular platelet clumping and thrombotic microangiopathy. Our patient, a 26-year-old man, had attacks of thrombotic thrombocytopenic purpura (TTP) with thrombocytopenia and a urine dipstick positive for hemoglobin (4+), often as the only sign of hemolytic activity. He had ADAMTS13 activity of <1% of normal plasma without the presence of inhibitors of ADAMTS13. ADAMTS13 deficiency was caused by two new mutations of the ADAMTS13 gene: a deletion of a single nucleotide in exon17 (c. 2042 delA) leading to a frameshift (K681C fs X16), and a missense mutation in exon 25 (c.3368G>A) leading to p.R1123H. This case report confirms the importance of the analysis of the ADAMTS13 activity and its inhibitor in patients who have episodes of TTP, with a very low platelet count and sometimes without the classic biochemical signs of hemolysis.

Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.