Markus Nagl

Molecular analysis of volatile metabolites released specifically by staphylococcus aureus and pseudomonas aeruginosa

BackgroundThe routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non- invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs).ResultsHeadspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole).Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml].ConclusionsThe results obtained provide strong evidence that the detection and perhaps even identification of bacteria could be achieved by determination of characteristic volatile metabolites, supporting the clinical use of breath-gas analysis as non-invasive method for early detection of bacterial lung infections.

Bactericidal activity of micromolar N-chlorotaurine: evidence for its antimicrobial function in the human defense system.

ABSTRACT N-Chlorotaurine, the main representative of long-lived oxidants found in the supernatant of stimulated granulocytes, has been investigated systematically with regard to its antibacterial activity at different physiological concentrations for the first time.N-Chlorotaurine (12.5 to 50 μM) demonstrated a bactericidal effect i.e., a 2 to 4 log10 reduction in viable counts, after incubation at 37°C for 6 to 9 h at pH 7.0, which effect was significantly enhanced in an acidic milieu (at pH 5.0), with a 3 to 4 log10 reduction after 2 to 3 h. Moreover, bacteria were attenuated after being incubated inN-chlorotaurine for a sublethal time, as demonstrated with the mouse peritonitis model. The supernatant of stimulated granulocytes exhibited similar activity. Transmission electron microscopy revealed changes in the bacterial cell membrane and cytoplasmic disintegration with both reacting systems, even in the case of a mere attenuation. The results of this study suggest a significant bactericidal function ofN-chlorotaurine and other chloramines during inflammation.

Chemical properties of N-chlorotaurine sodium, a key compound in the human defence system.

N‐Chlorotaurine (NCT) is known to play an important role in the human defence system. The already proved utility of the sodium salt as a disinfectant in human medicine suggested a thorough investigation of its chemical properties. Chlorine transfer to N‐H groups (transhalogenation) and oxidation of thio and aromatic compounds represent its main reactions. Auto‐chlorination causes disproportionation forming N, N‐dichlorotaurine (NDCT) with Kd = [NDCT][taurine]/fa[NCT]2aH+ = (4.5 ± 0.8) × 106, while the reaction with ammonium releasing NH2Cl is characterised by KNHCl2 = [NH2Cl][taurine]/[NCT][NH  4+ ]f  a2 = 0.02 ± 0.004.The verified unique stability and lowlevel reactivity of NCT are considered essential for its function in the mammalian defence system and its practical applicability, which manifests itself in an optimal compromise between microbicidal activity and toxicity.

Proposed nomenclature for Pseudallescheria, Scedosporium and related genera

As a result of fundamental changes in the International Code of Nomenclature on the use of separate names for sexual and asexual stages of fungi, generic names of many groups should be reconsidered. Members of the ECMM/ISHAM working group on Pseudallescheria/Scedosporium infections herein advocate a novel nomenclature for genera and species in Pseudallescheria, Scedosporium and allied taxa. The generic names Parascedosporium, Lomentospora, Petriella, Petriellopsis, and Scedosporium are proposed for a lineage within Microascaceae with mostly Scedosporium anamorphs producing slimy, annellidic conidia. Considering that Scedosporium has priority over Pseudallescheria and that Scedosporium prolificans is phylogenetically distinct from the other Scedosporium species, some name changes are proposed. Pseudallescheria minutispora and Petriellidium desertorum are renamed as Scedosporium minutisporum and S. desertorum, respectively. Scedosporium prolificans is renamed as Lomentospora prolificans.

Phagocytosis and Killing of Bacteria by Professional Phagocytes and Dendritic Cells

ABSTRACT Dendritic cells (DC) represent a class of professional antigen-presenting cells whose primary function is to alert the immune system, not to clear invading microorganisms. The objective of our study was to compare the abilities of polymorphonuclear neutrophilic leukocytes (PMN), monocytes, monocyte-derived macrophages (MDM), monocyte-derived immature DC (imDC), and mature DC (maDC) to ingest and destroy Staphylococcus aureus and Escherichia coli. Acridine orange staining and fluorescence microscopy demonstrated that MDM, followed by monocytes, imDC, and PMN, internalized bacteria well but that maDC exhibited less pronounced phagocytic activity. PMN, monocytes, and MDM exhibited a much higher capacity to kill ingested bacteria than both imDC and maDC. In summary, these data are in agreement with the generally accepted idea that different types of leukocytes fulfill specialized tasks in antigen presentation and killing of pathogens.

Studies of In Vitro Activities of Voriconazole and Itraconazole against Aspergillus Hyphae Using Viability Staining

ABSTRACT The minimal fungicidal concentrations (MFCs) of voriconazole and itraconazole for five clinical isolates each of Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger were determined by a broth macrodilution method. Conidial suspensions as inocula were compared to hyphae as inocula since the invasive form of aspergillosis is manifested by the appearance of hyphal structures. In addition, cell viability staining with the dye FUN-1 was performed to assess time-dependent damage of hyphae exposed to various concentrations of the antifungal agents. With conidial inocula the MFC ranges of voriconazole were 0.5 to 4 μg/ml and those of itraconazole were 0.25 to 2 μg/ml, whereas the MFCs (2 to >16 μg/ml) with hyphal inocula were substantially higher (P < 0.01) for both itraconazole and voriconazole. Only minor differences between the tested antifungals were observed since 16 of 20 and 17 of 20 of the isolates of Aspergillus spp. tested appeared to be killed by voriconazole and itraconazole, respectively. The results of FUN-1 viability staining correlated closely to colony counts, but various time- and dose-dependent levels of viability of hyphae were also observed. In conclusion, our study demonstrates the importance of the type of inoculum used to test antifungals and the applicability of FUN-1 staining as a rapid and sensitive method for assaying the viability of hyphae.

N-chlorotaurine, a natural antiseptic with outstanding tolerability

N-chlorotaurine, the N-chloro derivative of the amino acid taurine, is a long-lived oxidant produced by activated human granulocytes and monocytes. Supported by a high number of in vitro studies, it has mainly anti-inflammatory properties and seems to be involved in the termination of inflammation. The successful synthesis of the crystalline sodium salt (Cl-HN-CH(2)-CH(2)-SO(3)Na, NCT) facilitated its development as an endogenous antiseptic. NCT can be stored long-term at low temperatures, and it has killing activity against bacteria, fungi, viruses and parasites. Transfer of the active chlorine to amino groups of molecules of both the pathogens and the human body (transhalogenation) enhances rather than decreases its activity, mainly because of the formation of monochloramine. Furthermore, surface chlorination after sublethal incubation times in NCT leads to a post-antibiotic effect and loss of virulence of pathogens, as demonstrated for bacteria and yeasts. Being a mild oxidant, NCT proved to be very well tolerated by human tissue in Phase I and II clinical studies. A 1% aqueous solution can be applied to the eye, skin ulcerations, outer ear canal, nasal and paranasal sinuses, oral cavity and urinary bladder, and can probably be used for inhalation. Therapeutic efficacy in Phase II studies has been shown in external otitis, purulently coated crural ulcerations and keratoconjunctivitis, so far. Based upon all presently available data, NCT seems to be an antiseptic with a very good relation between tolerability and activity. Recently, C-methylated derivatives of NCT have been invented, which are of interest because of improved stability at room temperature.

Secretoneurin, an Angiogenic Neuropeptide, Induces Postnatal Vasculogenesis

Background—Induction of postnatal vasculogenesis, the mobilization of bone marrow–derived endothelial progenitor cells and incorporation of these cells into sites of blood vessel formation, is a well-known feature of angiogenic cytokines such as vascular endothelial growth factor. We hypothesized that the angiogenic neuropeptide secretoneurin induces this kind of neovascularization. Methods and Results—Secretoneurin induced mobilization of endothelial progenitor cells to sites of vasculogenesis in vivo in the cornea neovascularization assay. Progenitor cells were incorporated into vascular structures or were located adjacent to them. Systemic injection of secretoneurin led to increase of circulating stem cells and endothelial progenitor cells. In vitro secretoneurin induced migration, exerted antiapoptotic effects, and increased the number of these cells. Furthermore, secretoneurin stimulated the mitogen-activated protein kinase system, as shown by phosphorylation of extracellular signal–regulated kinase, and activated the protein kinase B/Akt pathway. Activation of mitogen-activated protein kinase was necessary for increase of cell number and migration, whereas Akt seemed to play a role in migration of endothelial progenitor cells. Conclusions—These data show that the angiogenic neuropeptide secretoneurin stimulates postnatal vasculogenesis by mobilization, migration, and incorporation of endothelial progenitor cells.

Characterization of volatile metabolites taken up by or released from Streptococcus pneumoniae and Haemophilus influenzae by using GC-MS.

Volatile organic compounds (VOCs) released from or taken up by Streptococcus pneumoniae and Haemophilus influenzae cultures were analysed by means of GC-MS after adsorption of headspace samples on multi-bed sorption tubes. Sampling was performed at different time points during cultivation of bacteria to follow the dynamics of VOC metabolism. VOCs were identified not only by spectral library match but also based on retention times of native standards. As many as 34 volatile metabolites were released from S. pneumoniae and 28 from H. influenzae, comprising alcohols, aldehydes, esters, hydrocarbons, ketones and sulfur-containing compounds. For both species, acetic acid, acetaldehyde, methyl methacrylate, 2,3-butanedione and methanethiol were found in strongly elevated concentrations and 1-butanol and butanal in moderately elevated concentrations. In addition, characteristic volatile biomarkers were detected for both bacterial species and exclusively for S. pneumoniae, also catabolism of aldehydes (3-methylbutanal and hexanal) was found. The results obtained provide important input into the knowledge about volatile bacterial biomarkers, which may become particularly important for detection of pathogens in upper airways by breath-gas analysis in the future.

Activity of N-chlorotaurine against herpes simplex- and adenoviruses

N-chlorotaurine, an essential weak oxidant produced by stimulated human leukocytes, is known to have bactericidal, fungicidal and vermicidal properties. This study for the first time demonstrates its virucidal activity. By viral suspension tests at incubation times between 5 and 60 min, virus titers of both Herpes simplex virus type 1 and 2 were reduced about 1.3-2.9 log10 and 2.8-4.2 log10 by 0.1 and 1%, (5.5 and 55 mM) N-chlorotaurine, respectively. Virus titer reduction of adenovirus type 5 between 15 and 60 min was 0.5-2.0 and 0.6-4.0 log10, respectively, by the same concentrations of N-chlorotaurine. These findings support a contribution of N-chlorotaurine in destruction of pathogens during inflammatory reactions and also the possibility of its application as an antiviral agent in human medicine.