A rapid method for breath analysis in cystic fibrosis patients.

For easy handling and speed of lung diseases diagnostics, approaches based on volatile organic compounds (VOCs), including those emitted by pathogenic microorganisms, are considered but currently require considerable sampling efforts. We tested whether easy-to-handle and fast detection of lung infections is possible using solid-phase microextraction (SPME) of 100 ml of exhaled breath. An analytical procedure for the detection of VOCs from the headspace of epithelial lung cells infected with four human pathogens was developed. The feasibility of this method was tested in a cystic fibrosis (CF) outpatient clinic in vivo. Exhaled breath was extracted by SPME and analyzed by gas chromatography-mass spectrometry (GC-MS). The compositions of VOCs released in the infection model were characteristic for all individual pathogens tested. Exhaled breath of CF patients allowed clear distinction of CF patients and controls by their VOC compositions using multivariate analyses. Interestingly, the major specific VOCs detected in the exhaled breath of infected CF patients in vivo differed from those monitored during bacterial in vitro growth. SPME extraction of VOCs from 100 ml of human breath allowed the distinction between CF patients and healthy probands. Our results highlight the importance of assessing the entire pattern of VOCs instead of selected biomarkers for diagnostic purposes, as well as the need to use clinical samples to identify reliable biomarkers. This study provides the proof-of-concept for the approach using the composition of exhaled VOCs in human breath for the rapid identification of infectious agents in patients with lower respiratory tract infections.

Microbial diversity of supra- and subgingival biofilms on freshly colonized titanium implant abutments in the human mouth.

Supra- and subgingival biofilm formation is considered to be mainly responsible for early implant failure caused by inflammations of periimplant tissues. Nevertheless, little is known about the complex microbial diversity and interindividual similarities around dental implants. An atraumatic assessment was made of the diversity of microbial communities around titanium implants by single strand conformation polymorphism (SSCP) analysis of the 16S rRNA gene amplicons as well as subsequent sequence analysis. Samples of adherent supra- and subgingival periimplant biofilms were collected from ten patients. Additionally, samples of sulcusfluid were taken at titanium implant abutments and remaining teeth. The bacteria in the samples were characterized by SSCP and sequence analysis. A high diversity of bacteria varying between patients and within one patient at different locations was found. Bacteria characteristic for sulcusfluid and supra- and subgingival biofilm communities were identified. Sulcusfluid of the abutments showed higher abundance of Streptococcus species than from residual teeth. Prevotella and Rothia species frequently reported from the oral cavity were not detected at the abutments suggesting a role as late colonizers. Different niches in the human mouth are characterized by specific groups of bacteria. Implant abutments are a very valuable approach to study dental biofilm development in vivo.

Kinetics of carbon sharing in a bacterial consortium revealed by combining stable isotope probing with fluorescence-activated cell sorting.

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 LD50 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.

Antibiofilm activity of Cobetia marina filtrate upon Staphylococcus epidermidis catheter-related isolates.

We report the antibiofilm activity by the sponge-associated bacterium Cobetia marina upon Staphylococcus epidermidis clinical isolates obtained from central venous catheters. Antibiofilm activity/antimicrobial susceptibility correlation might predict the action of the metabolite(s) upon Staphylococcus epidermidis in the clinic, making it a possible adjuvant in therapies against biofilm-associated infections.

In situ experimental evidence of the fate of a phytodetritus pulse at the abyssal sea floor.

More than 50% of the Earth' s surface is sea floor below 3,000 m of water. Most of this major reservoir in the global carbon cycle and final repository for anthropogenic wastes is characterized by severe food limitation. Phytodetritus is the major food source for abyssal benthic communities, and a large fraction of the annual food load can arrive in pulses within a few days. Owing to logistical constraints, the available data concerning the fate of such a pulse are scattered and often contradictory, hampering global carbon modelling and anthropogenic impact assessments. We quantified (over a period of 2.5 to 23 days) the response of an abyssal benthic community to a phytodetritus pulse, on the basis of 11 in situ experiments. Here we report that, in contrast to previous hypotheses, the sediment community oxygen consumption doubled immediately, and that macrofauna were very important for initial carbon degradation. The retarded response of bacteria and Foraminifera, the restriction of microbial carbon degradation to the sediment surface, and the low total carbon turnover distinguish abyssal from continental-slope 'deep-sea' sediments.

Novel glycine containing glucolipids from the alkane using bacterium Alcanivorax borkumensis.

The polar lipids from the hydrocarbon using and biosurfactant-producing bacterium Alcanivorax borkumensis were isolated and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The biosurfactant produced by this species is an anionic glucose lipid with a tetrameric oxyacyl side chain. The glycolipids extracted from the cell wall consist of this biosurfactant N-terminally esterified with glycine. Ten different derivatives of this lipid type were identified and their structures elucidated by MSMS. They vary by the chain length of one or two of the four beta-hydroxy fatty acids (C6, C8 and C10) and by the location of these different fatty acids within the molecule. All compounds are reported here for the first time. In addition to these glycolipids, three different phosphatidylglycerols were identified. While these lipids were found in all strains of A. borkumensis, the relative abundances of the different lipids vary between the strains.

Structural characterization of lichenysin A components by fast atom bombardment tandem mass spectrometry.

The structural characterization of the cyclic lipoheptapeptide surfactant lichenysin A components, produced by Bacillus licheniformis strains via the non-ribosomal pathway on a corresponding peptide synthetase, was carried out using a tandem mass spectrometry (MS/MS) under fast atom bombardment (FAB) conditions. Based on the analysis of the collision-induced fragment-ion spectrum of the single charged molecular ions of both native and partially hydrolyzed forms of lipopeptide, a new general structure of lichenysin A components was elucidated. It varies from previously proposed structure by having in the peptide portion of lipopeptide the L-Gln-1 and L-Asp-5 residues instead of L-Glu-1 and L-Asn-5. The verified chemical structure of lichenysin A was found to be reflected in the structural organization of the corresponding lichenysin A synthetase, LchA, described recently.

Bioactive sesquiterpenes produced by fungi: are they useful for humans as well?

Higher fungi are characterised by the production of macroscopic fruiting bodies to generate and to distribute their spores. These fruiting bodies are under constant threat of other organisms feeding on them. As a consequence these organisms developed a number of strategies for protection, one of them is the production of toxins. The fungal subdivision Basidiomycotina produce toxic sesquiterpenes many of them are derived from the protoilludane skeleton. This skeleton is transformed and rearranged to a large number of compounds. Some of these sesquiterpenes show interesting biological properties which may be attractive for medicinal chemistry. The overview describes the different types of bioactive fungal sesquiterpenes derived from humulene known to date in Basidiomycotina and their formation. The metabolites are discussed according to their sesquiterpene skeleton and the different metabolites are compared. Where available biological activities concerning antifungal, antibacterial, cytotoxic and enzyme inhibition data are given. Special attention was paid for the different activities of these metabolites and the attempts made to use them in medicinal chemistry. The question whether metabolites produced for the self-protection of fungi can be used for pharmaceutical applications for humans will be addressed and discussed.

Microbial reduction of aromatic carboxylic acids.

Several benzoic, cinnamic and phenylacetic acid derivatives were screened with 20 micro-organisms, mainly fungi, for the reduction of their carboxylic function. For all organisms several compounds were reduced in fairly good yields up to 80% to the corresponding alcohol. No general rule could be established, concerning the substitution pattern, as to which compounds were transformed to the alcohol. Generally the reactions were accomplished within 48-70 h. Only minor, if any, side products were detected. Dicarboxylic acids, such as phthalic or phenylglutaric acids and similar compounds could not be reduced by the microorganisms tested.

Antimicrobial and biofilm inhibiting diketopiperazines.

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.

Adenosine in the inflamed gut: a Janus faced compound.

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.

Antibiofilm activity of Cobetia marina filtrate upon Staphylococcus epidermidis catheter-related isolates

We report the antibiofilm activity by the sponge-associated bacterium Cobetia marina upon Staphylococcus epidermidis clinical isolates obtained from central venous catheters. Antibiofilm activity/antimicrobial susceptibility correlation might predict the action of the metabolite(s) upon Staphylococcus epidermidis in the clinic, making it a possible adjuvant in therapies against biofilm-associated infections.

Microbial formation of caparrapidiol and derivatives from trans-nerolidol.

Of 60 fungi and 40 bacteria, five strains, all belonging to the genus Fusarium, were able to hydrate (S)-trans-nerolidol to caparrapidiol. Fusarium moniliforme DSM 764 and F. tabacinum DSM 2125 oxidized trans-nerolidol to six new derivatives of caparrapidiol which were characterized. These products served to elucidate the absolute configuration of the naturally occurring caparrapidiol. Six further strains, again all Fusarium spp., hydrated geranylacetone at the inner double bond. The ecological implications of this rather rare biotransformation reaction are briefly discussed.

Phylogenetic influences in microbial hydroxylation of terpenoids.

Forty strains of bacteria and 60 of fungi were screened with globulol, 1,8-cineol and cedrol and the strains compared, on the basis of the resulting products, using multivariate analyses. The taxonomic position of the strains is mirrored in their biotransformation capability. Fungi and bacteria, fungal phyla, and Gram-positive and-negative bacteria could all be distinguished in a discriminant analysis using 12 substrates. Hierarchical clustering yielded five groups of strains with distinct biotransformation activity. Such clustering will allow a more efficient screening than before.

Ratios of carbon isotopes in microbial lipids as an indicator of substrate usage.

The occurrence and abundance of microbial fatty acids have been used for the identification of microorganisms in microbial communities. However, these fatty acids can also be used as indicators of substrate usage. For this, a systematic investigation of the discrimination of the stable carbon isotopes by different microorganisms is necessary. We grew 11 strains representing major bacterial and fungal species with four different isotopically defined carbon sources and determined the isotope ratios of fatty acids of different lipid fractions. A comparison of the differences of delta13C values of palmitic acid (C16:0) with the delta13C values of the substrates revealed that the isotope ratio is independent of the growth stage and that most microorganisms showed enrichment of C16:0 with 13C when growing on glycerol. With the exception of Burkholderia gladioli, all microorganism showed depletion of 13C in C16:0 while incorporating the carbons of glucose, and most of them were enriched with 13C from mannose, with the exception of Pseudomonas fluorescens and the Zygomycotina. Usually, the glycolipid fractions are depleted in 13C compared to the phospholipid fractions. The delta13C pattern was not uniform within the different fatty acids of a given microbial species. Generally, tetradecanoic acid (C14:0) was depleted of 13C compared to palmitic acid (C16:0) while octadecanoic acid (C18:0) was enriched. These results are important for the calibration of a new method in which delta13C values of fatty acids from the environment delineate the use of bacterial substrates in an ecosystem.

Intraspecific variation of unusual phospholipids from Corynebacterium spp. containing a novel fatty acid.

The novel fatty acid trans-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as Arthrobacter globiformis) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of Corynebacterium variabilis DSM 20132(T) and DSM 20536. All three strains possessed the novel fatty acid, identifying trans-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of Corynebacterium sp. strain LMG 3820 were similar to those of the type strain but different from those of Corynebacterium variabilis DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132(T) and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches.

Microbial transformations of some monoterpenoids and sesquiterpenoids.

The absolute configurations of fragrances, flavours and drugs are often important for their special properties. The growing interest of organic chemists in chiral synthons has stimulated work on biotransformations, for which readily available and inexpensive compounds can be used as substrates. Microbial transformations of 1-menthenes like gamma-terpinene, alpha-terpinene, limonene and alpha-phellandrene give the corresponding 1,2-trans-diols with high stereospecificity. Because of the volatility and toxicity of these substrates, and their low solubility in aqueous solutions, a special fermentation technique has been developed in which the terpenes are fed continuously to extended cultures of Corynespora cassiicola or Diplodia gossypina. (4R)-Limonene is transformed by Gibberella cyanea to (1S,2S,4R)-p-menth-8-en-1,2-diol, but 3,3,5,5-tetramethyl-limonene yields a 6-monohydroxylated product and a 6,10-dihydroxylated product with a 6-hydroxy-8,10-epoxy structure as the main metabolite. Vicinal diols are also formed from aliphatic terpenes, by reaction at the terminal isoprenoid groups. Some oxirane structures are found as intermediates. Acyclic sesquiterpenes often form complex mixtures when they are metabolized further. The products of the transformation of trans-nerolidol by several fungi are given as examples. Cyclic sesquiterpenes, with less flexible structures, are oxidized more specifically. Whereas longifolene is a very poor substrate for Corynespora cassiicola, isolongifolene is always hydroxylated at one of the methyl groups attached to C-7. The 14- or 15-hydroxy compounds are further oxidized, very fast, in the 3 position or 4 position.

'Clay hutches': a novel interaction between bacteria and clay minerals.

Biofilm formation on a low-energy substratum floating on the surface of a water column overlying a polychlorinated biphenyl (PCB)-contaminated sandy clay soil was followed by light and electron microscopy. The biofilms that developed consisted of a dense lawn of clay aggregates, each one of which contained one or more bacteria, phyllosilicates and grains of iron oxide material, all held together by bacterial extracellular polysaccharides (EPS). The clay leaflets were arranged in the form of 'houses of cards' and gave the aggregates the appearance of 'hutches' housing the bacteria. Interestingly, although the soil is poor in carbon, and the weakly bioavailable PCBs constitute the principal source of carbon in this system, the bacteria contained electron-transparent structures presumed to be carbon storage granules. These, and the EPS material present in the hutches, indicate that carbon is not limiting in this system and, as PCBs have been found associated with the clay mineral fraction of the floating substratum, the clay particles may serve as carbon shuttles. The interesting possibilities that the 'clay hutches' may represent a 'soil microhabitat', a 'minimal nutritional sphere' and an 'effective survival unit' for autochthonous bacteria are noted. The formation of clay hutches by bacteria would seem to merit further investigation, particularly regarding their roles in bacterial processes in soil and in geological processes.