• Applications and impacts of stable isotope probing for analysis of microbial interactions.

      Abraham, Wolf-Rainer; group of Chemical microbiology, Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2014-06)
      Probing the interactions between microbes and their environment with stable isotopes became a powerful technique over the last years. While quadruple mass spectrometry or isotope ratio mass spectrometry (IRMS) require at least 300,000 bacterial cells, analysis at the single-cell level is possible with secondary ion mass spectrometry (SIMS) or Raman microspectrometry. While SIMS needs enrichments of more than 0.1 and Raman microscopy of more than 25 at.-%, IRMS can deal with 0.0001 at.-%. To find out who eats what, one has to discern between the different species in a community. Several methods have been introduced to discern between the different taxa in microbial communities, e.g., by using fatty acids as biomarkers, density centrifugation of DNA/RNA, or fluorescent in situ hybridization (FISH) with phylogenetic probes. While the biomarker approach can be coupled with the high sensitivity of the IRMS, the DNA approach gives in general a better phylogenetic resolution of the metabolic active microbes. A combination of both is the separation via coupling of FISH-probes to magnetic beads or fluorescent assisted cell sorting (FACS) of stained cells leading to fractions which can be analyzed by IRMS. Applying these techniques over a time course can reveal the metabolic kinetics and food webs. In this review, the different methods are presented with examples and their advantages and disadvantages are discussed. An outlook on the combination of the various techniques and their applications in microbial ecology is given.
    • Stabile Isotope erlauben Einblicke in Bakteriengemeinschaften

      Abraham, Wolf-Rainer; Chemische Mikrobiology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany (2013-09-25)
    • Human β-Defensin 2 Induces Extracellular Accumulation of Adenosine in Escherichia coli.

      Estrela, Andreia Bergamo; Rohde, Manfred; Gutierrez, Maximiliano Gabriel; Molinari, Gabriella; Abraham, Wolf-Rainer; Chemical Microbiology. (2013-09)
      Human β-defensins are host defense peptides performing antimicrobial as well as immunomodulatory functions. The present study investigated whether treatment of Escherichia coli with human β-defensin 2 could generate extracellular molecules of relevance for immune regulation. Mass spectrometry analysis of bacterial supernatants detected the accumulation of purine nucleosides triggered by β-defensin 2 treatment. Other cationic antimicrobial peptides tested presented variable outcomes with regard to extracellular adenosine accumulation; human β-defensin 2 was the most efficient at inducing this response. Structural and biochemical evidence indicated that a mechanism other than plain lysis was involved in the observed phenomenon. By use of isotope ((13)C) labeling, extracellular adenosine was found to be derived from preexistent RNA, and a direct interaction between the peptide and bacterial nucleic acid was documented for the first time for β-defensin 2. Taken together, the data suggest that defensin activity on a bacterial target may alter local levels of adenosine, a well-known immunomodulator influencing inflammatory processes.
    • Cauliform bacteria lacking phospholipids from an abyssal hydrothermal vent: proposal of Glycocaulis abyssi gen. nov., sp. nov., belonging to the family Hyphomonadaceae.

      Abraham, Wolf-Rainer; Lünsdorf, Heinrich; Vancanneyt, Marc; Smit, John; Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. wolf-rainer.abraham@helmholtz-hzi.de (2013-06)
      Cauliform bacteria are prosthecate bacteria often specialized for oligotrophic environments. A polyphasic approach, comprising 16S rRNA gene sequencing, lipid analysis and salt tolerance characterizations, was used to clarify the taxonomy of one isolate, strain MCS 33(T), obtained from above the hot water plume of a deep-sea hydrothermal vent near Vancouver island, Canada. Cells contained no detectable phospholipids or sulpholipids, but did contain 1,2-di-O-acyl-3-O-α-D-glucopyranosylglycerol, 1,2-di-O-acyl-3-O-α-D-glucopyranuronosylglycerol and the novel lipid 1,2-di-O-acyl-3-[O-α-D-glucopyranuronosyl]glycerol-6'-N-glycine. It is assumed that the various glucoronosyl lipids are replacing, at least partially, the phospholipids in their various tasks in the cell cycle. The G+C content of the genomic DNA of strain MCS 33(T) was 62.8 mol%, and Q10 was the predominant respiratory ubiquinone. The 16S rRNA gene sequence of this chemoheterotrophic, aerobic, moderately halophilic strain showed only a low similarity of 94.4% to that of Oceanicaulis alexandrii C116-18(T), and both strains also differed based on their lipids. Although the novel strain was isolated from seawater sampled near a hydrothermal vent, its optimum temperature for growth was 30 °C. The main cellular fatty acids were C18:1ω7c, C18:0 and the unknown fatty acid ECL 11.798, and the main hydroxy fatty acid was C12:0 3-OH. The strain is proposed to represent a novel species of a new genus, Glycocaulis abyssi gen. nov., sp. nov. The type strain of the type species is MCS 33(T) (=LMG 27140(T)=CCUG 62981(T)).
    • Dipeptide cis-cyclo(Leucyl-Tyrosyl) produced by sponge associated Penicillium sp. F37 inhibits biofilm formation of the pathogenic Staphylococcus epidermidis.

      Scopel, Marina; Abraham, Wolf-Rainer; Henriques, Amélia T; Macedo, Alexandre J; Faculdade de Farmácia, Departamento de Produção de Matéria-Prima, Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752, 90610-000 Porto Alegre, Brazil. (2013-02-01)
      Infections associated to microbial biofilms are involved in 80% of human infections and became a challenge concerning public health. Infections related to Staphylococcus epidermidis biofilms are presently commonly associated to medical devices, increasing treatment costs for this type of infection. Alternatives to eliminate this kind of disease have been employed in screening programs using diverse marine-derived fungi source of bioactive compounds capable to combat biofilm formation. In this work was isolated the dipeptide cis-cyclo(Leucyl-Tyrosyl) from a sponge associated Penicillium sp. possessing a remarkable inhibition up to 85% of biofilm formation without interfering with bacterial growth, confirmed by scanning electron microscopy. This is the first demonstration that cis-cyclo(Leucyl-Tyrosyl) is able to specifically inhibit biofilm formation adding another aspect to the broad spectrum of bioactivities of cyclic dipeptides.
    • Chemische Funktionalisierung und Materialoptimierung dentaler Implantat-Abutments zur Reduktion der oralen Biofilmbildung

      Stiesch, Meike; Menzel, Henning; Abraham, Wolf-Rainer; Müller, Peter Paul; Dempwolf, Wiebke; Pfaffenroth, Cornelia; Kohorst, Phillip; Winkel, Andreas; Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany. (Druckerei der Medizinischen Hochschule, 2012-07-06)
    • Antimicrobial and biofilm inhibiting diketopiperazines.

      de Carvalho, M P; Abraham, Wolf-Rainer; Helmholtz Center for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 Braunschweig, Germany. wolf-rainer.abraham@helmholtz-hzi.de. (2012-07-01)
      Diketopiperazines are the smallest cyclic peptides known. 90% of Gram-negative bacteria produce diketopiperazines and they have also been isolated from Gram-positive bacteria, fungi and higher organisms. Biosynthesis of cyclodipeptides can be achieved by dedicated nonribosomal peptide synthetases or by a novel type of synthetases named cyclopeptide synthases. Since the first report in 1924 a large number of bioactive diketopiperazines was discovered spanning activities as antitumor, antiviral, antifungal, antibacterial, antiprion, antihyperglycemic or glycosidase inhibitor agents. As infections are of increasing concern for human health and resistances against existing antibiotics are growing this review focuses on the antimicrobial activities of diketopiperazines. The antibiotic bicyclomycin is a diketopiperazine and structure activity studies revealed the unique nature of this compound which was finally developed for clinical applications. The antimicrobial activities of a number of other diketopiperazines along with structure activity relationships are discussed. Here a special focus is on the activity-toxicity problem of many compounds setting tight limitations to their application as drugs. Not only these classical antimicrobial activities but also proposed action in modulating bacterial communication as a new target to control biofilms will be evaluated. Pathogens organized in biofilms are difficult to eradicate because of the increase of their tolerance for antibiotics for several orders. Diketopiperazines were reported to modulate LuxR-mediated quorum-sensing systems of bacteria, and they are considered to influence cell-cell signaling offering alternative ways of biofilm control by interfering with microbial communication. Concluding the review we will finally discuss the potential of diketopiperazines in the clinic to erase biofilm infections.
    • Volatile sesquiterpenes from fungi: what are they good for?

      Kramer, Rolf; Abraham, Wolf-Rainer; Helmholtz Center for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 Braunschweig, Germany. (2012-03-05)
    • Kinetics of carbon sharing in a bacterial consortium revealed by combining stable isotope probing with fluorescence-activated cell sorting.

      Pawelczyk, S; Bumann, D; Abraham, Wolf-Rainer; Helmholtz Center for Infection Research, Chemical Microbiology, Braunschweig, Germany. (2011-02-07)
      Aims:  To determine the kinetics of substrate fluxes in a microbial community in order to elucidate the roles of the community members. Methods and Results:  The kinetics of substrate sharing in a bacterial consortium were measured by a new analytical approach combining immunostaining, stable isotope probing and fluorescence-activated cell sorting (FACS). The bacterial consortium, consisting of four strains and growing on 4-chlorosalicylate (4-CS), was pulse-dosed with the degradation intermediate [U-(13) C]-4-chlorocatechol (4-CC). Cells were stained with strain-specific antibodies sorted by FACS and the (13) C-incorporation into fatty acids of the two most abundant members of the community was determined by isotope ratio mass spectrometry. From the two most abundant strains, the primary degrader Pseudomonas reinekei MT1 incorporated the labelled substrate faster than strain Achromobacter spanius MT3 but the maximal incorporation in strain MT3 was almost three times higher than in MT1. Conclusions:  It has been reported that strain MT1 produces 4-CC as an intermediate but has a lower LD(50) for it than strain MT3; therefore, MT3 still degrades 4-CC when the concentrations of 4-CC are already too toxic, even lethal, for MT1. By degrading 4-CC, produced by MT1, MT3 protects the entire community against this toxin. The higher affinity but lower tolerance of strain MT1 for 4-chlorocatechol compared to strain MT3 explains the complementary function these two strains have in the consortium adding exceptional stability to the entire community. Significance and Impact of the Study:  The novel approach can reveal carbon fluxes in microbial communities generating quantitative data for systems biology of the microbial community.
    • Megacities as sources for pathogenic bacteria in rivers and their fate downstream.

      Abraham, Wolf-Rainer; Helmholtz Center for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 Braunschweig, Germany. (2011)
      Poor sanitation, poor treatments of waste water, as well as catastrophic floods introduce pathogenic bacteria into rivers, infecting and killing many people. The goal of clean water for everyone has to be achieved with a still growing human population and their rapid concentration in large cities, often megacities. How long introduced pathogens survive in rivers and what their niches are remain poorly known but essential to control water-borne diseases in megacities. Biofilms are often niches for various pathogens because they possess high resistances against environmental stress. They also facilitate gene transfers of antibiotic resistance genes which become an increasing health problem. Beside biofilms, amoebae are carriers of pathogenic bacteria and niches for their survival. An overview about our current understanding of the fate and niches of pathogens in rivers, the multitude of microbial community interactions, and the impact of severe flooding, a prerequisite to control pathogens in polluted rivers, is given.
    • Adenosine in the inflamed gut: a Janus faced compound.

      Estrela, A B; Abraham, Wolf-Rainer; Helmholtz Center for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 Braunschweig, Germany. (2011)
      The purine ribonucleoside adenosine (Ado) has been recognized for its regulatory functions in situations of cellular stress like ischemia, hypoxia and inflammation. The importance of extracellular Ado as a modulator in the immune system is a theme of great appreciation and the focus of recent increasing interest in the field of gastrointestinal inflammation. In this review, the different aspects of Ado signaling during inflammatory responses in the gut are discussed, considering the contribution of the four known Ado receptors (ARs; A(1), A(2A), A(2B), and A(3)), their mechanisms and expression patterns. Activation of these receptors in epithelial cells as well as in immune cells recruited to the inflamed intestinal mucosa determines the overall effect, ranging from a protective, anti-inflammatory modulation to a strong pro-inflammatory induction. Here we present the current advances in agonists and antagonists development and their potential therapeutic application studied in animal models of intestinal inflammation. In addition, alternative complementary approaches to manipulate such a complex signaling system are discussed, for example, the use of AR allosteric modulators or interference with Ado metabolism. Special features of the gut environment are taken into account: the contribution of diet components; the involvement of Ado in intestinal infections; the interactions with the gut microbiome, particularly, the recent exciting finding that an intestinal bacterium can directly produce extracellular Ado in response to host defense mechanisms in an inflammation scenario. Understanding each component of this dynamic system will broaden the possibilities for applying Ado signaling as a therapeutic target in gut inflammation.
    • Brevundimonas vancanneytii sp. nov., isolated from blood of a patient with endocarditis.

      Estrela, Andréia B; Abraham, Wolf-Rainer; Helmholtz Center for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 Braunschweig, Germany. (2010-09)
      A Gram-negative, rod-shaped, non-spore-forming bacterial strain, designated LMG 2337(T), was isolated from the blood of a patient with endocarditis and characterized. The strain was affiliated with the alphaproteobacterial genus Brevundimonas, with Brevundimonas diminuta LMG 2089(T) (98.3 % 16S rRNA gene sequence similarity) and Brevundimonas terrae KSL-145(T) (97.5 %) as its closest relatives. This affiliation was supported by chemotaxonomic data: the G+C content was 66.3 mol %, the major polar lipids were phosphatidyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidyl glucopyranosyl diacylglycerol and the major fatty acids were summed feature 7 (one or more of C(18 : 1)ω 7c, C(18 : 1)ω 9t and C(18 : 1)ω 12t) and C(16 : 0). Strain LMG 2337(T) displayed an unusually broad substrate spectrum. The results from DNA-DNA hybridization and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain LMG 2337(T) from all of the type strains of hitherto-described Brevundimonas species. The strain therefore represents a novel species, for which the name Brevundimonas vancanneytii sp. nov. is proposed, with type strain LMG 2337(T) (=CCUG 1797(T) =ATCC 14736(T)).
    • Combining Biofilm-Controlling Compounds and Antibiotics as a Promising New Way to Control Biofilm Infections

      Estrela, Andréia Bergamo; Abraham, Wolf-Rainer; HelmholtzCenter for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany (2010-05-21)
    • Combining Biofilm-Controlling Compounds and Antibiotics as a Promising New Way to Control Biofilm Infections.

      Estrela, Andréia Bergamo; Abraham, Wolf-Rainer; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2010-05-11)
      Many bacteria grow on surfaces forming biofilms. In this structure, they are well protected and often high dosages of antibiotics cannot clear infectious biofilms. The formation and stabilization of biofilms are mediated by diffusible autoinducers (e.g. N-acyl homoserine lactones, small peptides, furanosyl borate diester). Metabolites interfering with this process have been identified in plants, animals and microbes, and synthetic analogues are known. Additionally, this seems to be not the only way to control biofilms. Enzymes capable of cleaving essential components of the biofilm matrix, e.g. polysaccharides or extracellular DNA, and thus weakening the biofilm architecture have been identified. Bacteria also have mechanisms to dissolve their biofilms and return to planktonic lifestyle. Only a few compounds responsible for the signalling of these processes are known, but they may open a completely novel line of biofilm control. All these approaches lead to the destruction of the biofilm but not the killing of the pathogens. Therefore, a combination of biofilm-destroying compounds and antibiotics to handle biofilm infections is proposed. In this article, different approaches to combine biofilm-controlling compounds and antibiotics to fight biofilm infections are discussed, as well as the balance between biofilm formation and virulence.
    • Tree species affect atmospheric CH4 oxidation without altering community composition of soil methanotrophs

      Menyailo, Oleg V.; Abraham, Wolf-Rainer; Conrad, Ralf; Helmholtz Center for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany (Elsevier, 2010-01-01)
    • Community-based degradation of 4-chorosalicylate tracked on the single cell level.

      Pawelczyk, Sonja; Abraham, Wolf-Rainer; Harms, Hauke; Müller, Susann; University of Oxford, Department of Biochemistry, South Parks Road, OX1 3QU, Oxford, UK. (2008-09)
      4-Chlorosalicylate (4-CS) can be degraded completely by a bacterial consortium consisting of Pseudomonas reinekei (MT1), Achromobacter spanius (MT3) and Pseudomonas veronii (MT4). The fourth species Wautersiella falsenii (MT2) is thought to act as a 'necrotizer' of the community. Single cell approaches were used to follow every species' degradation activity within the community by assuming that growth and proliferation are activity markers for the utilization of 4-CS and its degradation pathway intermediates as carbon and energy sources. A primary/secondary antibody staining technique for species differentiation was applied and a species-resolved determination of proliferation activity by flow cytometry undertaken. Degradation was followed by quantifying 4-CS and the resulting intermediates by HPLC. A good correlation of HPLC bulk data with the proliferation activity states of every species within the community was found. It was also assumed that reduced activity of strain MT4 and increased proliferation of strain MT2 might have caused an observed breakdown of the consortium grown in the bioreactor. The double staining technique provided the chance to follow bacterial cell states and their roles in mixed cultures without applying labelled substrates. It is therefore in line with single cell techniques already successfully applied in biotechnology for developing strategies to optimize microbially catalyzed production processes.
    • Phylogeny by a polyphasic approach of the order Caulobacterales, proposal of Caulobacter mirabilis sp. nov., Phenylobacterium haematophilum sp. nov. and Phenylobacterium conjunctum sp. nov., and emendation of the genus Phenylobacterium.

      Abraham, Wolf-Rainer; Macedo, Alexandre J; Lünsdorf, Heinrich; Fischer, Roman; Pawelczyk, Sonja; Smit, John; Vancanneyt, Marc; Helmholtz Center for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, Braunschweig, Germany. wab@gbf.de (2008-08)
      Three strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from fresh water and human blood. As determined by analyses of 16S rRNA gene sequences, the prosthecate strain FWC 38T was affiliated to the alphaproteobacterial genus Caulobacter, with Caulobacter henricii (96.8 %) and Caulobacter fusiformis (96.8 %) as its closest relatives. The non-prosthecate strain LMG 11050T and the prosthecate strain FWC 21T both belonged to the genus Phenylobacterium with Phenylobacterium koreense (96.9 %) and Phenylobacterium immobile (96.3 %) as the closest relatives. This affiliation was supported by chemotaxonomic data (polar lipids and cellular fatty acids). Physiological and biochemical tests allowed genotypic and phenotypic differentiation of the novel strains from all hitherto recognized species of the genera Caulobacter and Phenylobacterium. The strains therefore represent novel species, for which the names Caulobacter mirabilis sp. nov. (type strain FWC 38T=LMG 24261T=CCUG 55073T), Phenylobacterium conjunctum (type strain FWC 21T=LMG 24262T=CCUG 55074T), the first described prosthecate Phenylobacterium species, and Phenylobacterium haematophilum sp. nov. (type strain LMG 11050T=CCUG 26751T) are proposed. Marker nucleotides within the 16S rRNA genes were determined for the genera Asticcacaulis, Brevundimonas, Caulobacter and Phenylobacterium and the description of the genus Phenylobacterium is emended.
    • Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids.

      Shrestha, Minita; Abraham, Wolf-Rainer; Shrestha, Pravin Malla; Noll, Matthias; Conrad, Ralf; Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, D-35043, Marburg, Germany. (2008-02)
      Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH(4) into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH(4) flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using (13)C-labelled CH(4) directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to (13)CH(4) for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.
    • Occurence and resistance of pathogenic bacteria along the Tiete river downstream of Soa Paulo in Brazil

      Abraham, Wolf-Rainer; Macedo, Alexandre Jose; Gomez, Luiz Humberto; Tavarez, Flavio C. A.; Helmholtz Center for Infection Research (2007-09-24)
    • Sphingomonas fennica sp. nov. and Sphingomonas haloaromaticamans sp. nov., outliers of the genus Sphingomonas.

      Wittich, Rolf-Michael; Busse, Hans-Jürgen; Kämpfer, Peter; Macedo, Alexandre J; Tiirola, Marja; Wieser, Monika; Abraham, Wolf-Rainer; Bereich Mikrobiologie, Helmholtz Zentrum für Infektionsforschung (formerly GBF), D-38124 Braunschweig, Germany. (2007-08)
      Bacterial isolates obtained from polychlorophenol-contaminated sites in Finland (strain K101(T)) and from a Dutch drinking water well (strain A175(T)) were characterized taxonomically. 16S rRNA gene sequence analysis, determination of DNA G+C content, physiological characterization, estimation of the ubiquinone and polar lipid patterns and fatty acid content revealed that strains K101(T) and A175(T) were similar to Sphingomonas wittichii RW1(T) but also showed pronounced differences. The DNA G+C contents of the two novel strains were 63.6 and 66.1 mol%, respectively. On the basis of these results, two novel species of the genus Sphingomonas are described, for which the names Sphingomonas haloaromaticamans sp. nov. [type strain A175(T) (=DSM 13477(T)=CCUG 53463(T))] and Sphingomonas fennica sp. nov. [type strain K101(T) (=DSM 13665(T)=CCUG 53462(T))] are proposed.