Bacterial quorum sensing molecule induces chemotaxis of human neutrophils via induction of p38 and leukocyte specific protein 1 (LSP1).

When bacteria colonize surfaces, they socialize and form biofilms. This process is well regulated and relies on the communication among the bacteria via so-called "quorum sensing molecules". Among those, N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12), generated by Pseudomonas aeruginosa and other Gram-negative bacteria, activates not only bacteria but also interacts with mammalian cells. Among others, it activates phagocytic cells and - as we had shown previously - it is chemotactic for human polymorphonuclear neutrophils (PMN) in vitro. In the present study, we analyzed the signalling pathway of AHL-12 in PMN. We focused on the mitogen activated protein (MAP) kinase p38, because SB203580, an inhibitor of p38, prevented the AHL-12 induced chemotaxis. We found that in response to AHL-12, p38 was phosphorylated within minutes, as was its downstream target, the MAPKAP-Kinase-2 (MK2). In PMN, the major substrate of MK2 is the leukocyte specific protein 1 (LSP1), which binds to F-actin and participates directly in actin polymerization and cell migration. In response to AHL-12, LSP1 was phosphorylated and co-localized with F-actin in polarized PMN, suggesting that AHL-12-induced migration depended on p38 and LSP1 activation.

The extracellular polymer substance of Pseudomonas aeruginosa: too slippery for neutrophils to migrate on?

PURPOSE: Biofilm formation is increasingly recognized as the cause of persistent infections and there is evidence that P. aeruginosa organized into biofilms are quite resistant toward host defence mechanisms, particularly against an attack by polymorphonuclear neutrophils (PMN). Apparently, the migration of PMN through the biofilms is impaired, and thus the bactericidal activity remains highly localized. The aim of this study was to directly investigate the interaction of PMN with the biofilm and the extracted extracellular polymeric substance (EPS) of P. aeruginosa. MATERIAL AND METHODS: Chemotaxis and random migration of PMN through P. aeruginosa biofilms was tested, as was their migration through and along the EPS. RESULTS: We found that the EPS and mature biofilms, but not immature or developing ones, reduced the chemotactic migration of PMN. On EPS, rather than immobilize the cells, their random, spontaneous migration was enhanced. CONCLUSION: We propose that on EPS, the PMN lose their capacity to sense the direction and just slide over the EPS in a disoriented manner.

Combating implant infections. Remarks by a women's team.

Research on implant infections requires cooperative efforts and integration between basic and clinical expertises. An international group of women scientists is acting together in this field. The main research topics of the participants of this group are described. Formation of bacterial biofilms, antibiotic resistance and production of virulence factors like adhesins and toxins are investigated. New biomaterials, coatings and drugs designed to inhibit microbial adhesion are evaluated, and infection-resistant biomaterials are under study, such as a novel heparinizable polycarbonate-urethane (Bionate) or incorporation of diamino-diamide-diol (PIME) to reduce bacterial attachment. The correlation between biofilm production and the accessory-gene-regulator (agr) is investigated in Staphylococcus aureus. The ability to form biofilm has also been shown to be one of the important virulence factors of Enterococcus faecalis, favouring colonization of inert and biological surfaces. The study of quorum sensing has led to the discovery of a quorum sensing inhibitor termed RIP that suppresses staphylococcal biofilm and infections. The immune response and the local defence mechanisms of the host against implant-associated infections, activation and infiltration of immunocompetent cells into the sites of infection have been studied in patients with implant-associated osteomyelitis. Production of monoclonal antibodies (mAbs) as possible vaccines against the staphylococcal collagen-binding MSCRAMMs is in progress.

Molecular monitoring of inactivation efficiencies of bacteria during pulsed electric field treatment of clinical wastewater.

AIMS: The applicability of an alternative wastewater disinfection concept based on the pulsed electric field (PEF) treatment is tested with molecular biology techniques using clinical wastewaters. METHODS AND RESULTS: Hospital wastewater was treated with the PEF technology. The inactivation efficiencies of bacteria were successfully monitored with real-time polymerase chain reaction (PCR). As the differentiation between living and dead bacterial cells is important for the determination of the disinfection efficiency, propidium monoazide (PMA) was applied. PMA selectively penetrates cells with compromised membranes and intercalates into the DNA inhibiting a subsequent PCR amplification. The rates of reduction were examined for specific pathogens and wastewater populations using PCR-denaturing gradient gel electrophoresis. The results showed that the main part of the bacterial population could be inactivated efficiently with the PEF treatment. Moreover, it was demonstrated that naturally occurring nuclease activities were not affected by the PEF treatment in contrast to a thermal treatment. CONCLUSIONS: The results indicated that the PEF treatment is an appropriate alternative disinfection concept for the treatment of clinical wastewaters and surpass the disadvantages of other disinfection methods. SIGNIFICANCE AND IMPACT OF THE STUDY: With the use of propidium monoazide for live-dead distinction, a new concept could be developed for the evaluation of disinfection methods.

Antibiotic resistant pathogenic bacteria and their resistance genes in bacterial biofilms.

Biofilm-forming bacteria are ubiquitous in the environment and also include biofilm-forming pathogens. Environmental biofilms may form a reservoir for risk genes and may act as a challenge for human health. Examples of the health relevance of biofilms are the increase in antibiotic resistant bacteria hosted in biofilms in hospital and environment and consequently the interaction of these bacteria with human cells, e.g. in the immune system. Although data concerning the occurrence and spread of resistant bacteria within hospital care units are available, the fate of these bacteria in the environment and especially in the aquatic environment has barely been investigated. Once antibiotic resistant bacteria have entered the environment, a back coupling by ingestion or other possible entry into the host has to be prevented. Therefore a strategy to investigate paths of entry, accumulation and spread of resistant bacteria in environmental compartments has been developed using quantitative determination of genetic resistance determinants. Additionally a bacterial bioassay assessed bioeffectivity thresholds of low antibiotic concentrations. This approach enables an evaluation of the potential of contaminated waters to exert a selection pressure on bacterial communities and thus promote the persistence of resistant organisms. Completed with an indicator system for the identification of sources of multiresistant bacteria a concept for monitoring and evaluation of environmental compartments with respect to their potential of antibiotic resistance dissemination is suggested.

Implant-associated posttraumatic osteomyelitis: collateral damage by local host defense?

Infections following osteosynthesis or total joint replacement, also known as ''implant-associated posttraumatic osteomyelitis'', represent a major complication in orthopedic and trauma surgery. While the formation of bacterial biofilms on the implanted osteosynthesis materials is generally accepted as cause of the persistent infection, the molecular mechanisms leading to the progressive and destructive local inflammatory process and eventually to bone degradation, the osteolysis, have not been delineated. Here we provide evidence supporting the hypothesis that it is not the infection per se that causes tissue degradation and osteolysis, but rather the cytotoxic, proteolytic, and proinflammatory effector functions of cells of the host defense, particularly of the infiltrating polymorphonuclear neutrophils.

Formation of natural biofilms during chlorine dioxide and u.v. disinfection in a public drinking water distribution system.

AIMS: The influence of two disinfection techniques on natural biofilm development during drinking water treatment and subsequent distribution is compared with regard to the supply of a high-quality drinking water. METHODS AND RESULTS: The growth of biofilms was studied using the biofilm device technique in a real public technical drinking water asset. Different pipe materials which are commonly used in drinking water facilities (hardened polyethylene, polyvinyl chloride, steel and copper) were used as substrates for biofilm formation. Apart from young biofilms, several months old biofilms were compared in terms of material dependence, biomass and physiological state. Vital staining of biofilms with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA-specific 4',6-diamidino-2-phenylindole (DAPI) staining resulted in a significant difference in physiological behaviour of biofilm populations depending on the disinfection technique. Compared with chlorine dioxide disinfection (0.12-0.16 mg l-1), the respiratory activities of the micro-organisms were increased on all materials during u.v. disinfection (u.v.254; 400 J m-2). The biofilm biocoenosis was analysed by in situ hybridization with labelled oligonucleotides specific for some subclasses of Proteobacteria. Using PCR and additional hybridization techniques, the biofilms were also tested for the presence of Legionella spp., atypical mycobacteria and enterococci. The results of the molecular-biological experiments in combination with cultivation tests showed that enterococci were able to pass the u.v. disinfection barrier and persist in biofilms of the distribution system, but not after chlorine dioxide disinfection. CONCLUSIONS: The results indicated that bacteria are able to regenerate and proliferate more effectively after u.v. irradiation at the waterworks, and chlorine dioxide disinfection appears to be more applicative to maintain a biological stable drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as the application of u.v. disinfection is used for conditioning of critical water sources for drinking water, the efficiency of u.v. irradiation in natural systems should reach a high standard to avoid adverse impacts on human health.

A comparison of five rapid direct toxicity assessment methods to determine toxicity of pollutants to activated sludge.

Five rapid direct toxicity assessment methods were used in three European partner countries to determine the toxicity of single toxicants, mixed toxicants and real industrial wastes. The final aim was to protect microbial degradation of organic wastes in biological treatment processes and hence enhance the quality of treated effluents to be discharged to the environment. Nitrification inhibition, Respirometry, Adenosine triphosphate luminescence and Enzyme inhibition were tested utilising activated sludge as the testing matrix. The Vibrio fischeri toxicity test was used as a surrogate to compare the various microbial bioassays. The IC50 (toxicant concentration eliciting a 50% inhibitory effect) was determined for a number of pollutants including single toxicants Cd, Cr, Cu, Zn, 3,5-dichlorophenol, toluene and linear alkylbenzenesulphonate (LAS); a standard mixture of metals and LAS; a standard mixture of organics and LAS, and 16 industrial effluents. The V. fischeri bioassay was also chosen in order to assess quality control of toxicant preparation during testing in the different laboratories of the partner countries. Comparisons of sensitivity, cost of implementation, cost per test, relevance, and ease of use were made. The most sensitive bioassays were V. fischeri and Nitrification inhibition, however, this depended in the main on the pollutant and mixtures tested. It is recommended that during assessment of wastewater toxicity a suite of tests be used rather than reliance on one particular test.

Rapid parallel detection of hygienically relevant microorganisms in water samples by PCR and specific hybridization in microtiter plates.

A molecular biological test protocol for the parallel detection of enterococci and Pseudomonas aeruginosa in drinking water was developed. Amplicons labelled with digoxigenin during PCR were hybridized to specific 23S rDNA targeted oligonucleotide probes immobilized in microtiter plates. Detection was performed by addition of anti-digoxigenin-peroxidase-conjugate and chromogenic substrate. Specificity of the probes was evaluated by using pure cultures. First evaluation data with natural water samples in comparison to conventional microbiological analysis according to the German Drinking Water Regulation showed good agreement. Its feasible and rapid performance should be advantageous for use in routine drinking water quality control. Further comparative evaluation studies need to be undertaken to determine the true applicability for routine testing of water samples.

A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization.

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.

Application of 23S rDNA-targeted oligonucleotide probes specific for enterococci to water hygiene control.

Identification of enterococci species by hybridization with recently designed species-specific and group-specific 23S rDNA-targeted oligonucleotide probes was superior to results obtained with a common biochemical test panel. Considering these findings, a molecular biological procedure for the detection of enterococci in water samples was developed. A short enrichment is followed by an amplification step and a hybridization reaction in microtiter plate format. The detection limit is about 1 CFU/ml, and results are available within 26 h.

Molecular assembly and encapsulation directed by hydrogen-bonding preferences and the filling of space.

Multiple copies of a molecule, held together in finite aggregates, give rise to properties and functions that are unique to their assembled states. Because these aggregates are held together by weak forces operating over short distances, a premium is placed on complementarity: The molecular surfaces must facilitate specific interactions that direct the assembly to one aggregate rather than another. Hydrogen-bonding preferences can be combined with molecular curvature to favor the assembly of four self-complementary subunits into a pseudo-spherical capsule. Filling the capsule with smaller, complementary molecules provides the final instruction for the assembly process.

[Comparison of four methods for the detection of fecal streptococci in water]. / Vergleich von vier Nachweismethoden für Fäkalstreptokokken in Wasser.

The objective of this study was to select an effective and fast method for the detection of socalled fecal streptococci in water by comparing a method according to the German drinking water standard, a membrane filtration method according to the ISO-draft standard 7899/2, the Chromocult Enterococcus Broth (Merck) and the Enterolert-System (IDEXX). The study was based on a collective of 297 water samples derived from different stages of water treatment and distribution, as well as from individual water supplies. The sensitivity, reliability, and selectivity of the single methods in relation to their practicability was evaluated. Concerning false positive and false negative results, the tests were proved by metabolic characterization of the isolated strains. The advantages and disadvantages of the methods resulting from the investigated criteria are discussed. The work is part of a comparative study within the scope of the DIN ad-hoc-committee "fecal streptococci".

Molecular recognition at the thrombin active site: structure-based design and synthesis of potent and selective thrombin inhibitors and the X-ray crystal structures of two thrombin-inhibitor complexes.

BACKGROUND: The serine protease thrombin is central in the processes of hemostasis and thrombosis. To be useful, thrombin inhibitors should combine potency towards thrombin with selectivity towards other related enzymes such as trypsin. We previously reported the structure-based design of thrombin inhibitors with rigid, bicyclic core structures. These compounds were highly active towards thrombin, but showed only modest selectivity. RESULTS: Here, we describe the rational design of selective thrombin inhibitors starting from the X-ray crystal structure of the complex between the previously generated lead molecule and thrombin. The lead molecule bound with a Ki value of 90nM and a selectivity of 7.8 for thrombin over trypsin. Our design led to inhibitors with improved activity and greatly enhanced selectivity. The binding mode for two of the new inhibitors was determined by X-ray crystallography of their complexes with thrombin. The results confirmed the structures predicted by molecular modeling and, together with the binding assays, provided profound insight into molecular recognition phenomena at the thrombin active site. CONCLUSIONS: A novel class of nonpeptidic, selective thrombin inhibitors has resulted from structure-based design and subsequent improvement of the initial lead molecule. These compounds, which are preorganized for binding to thrombin through a rigid, bicyclic or tricyclic central core, could aid in the development of new antithrombotic drugs. Correlative binding and X-ray structural studies within a series of related, highly preorganized inhibitors, which all prefer similar modes of association to thrombin, generate detailed information on the strength of individual intermolecular bonding interactions and their contribution to the overall free energy of complexation.

Detection of primary DNA damage in Chlamydomonas reinhardtii by means of modified microgel electrophoresis.

The assessment of genotoxic potential in surface water requires test methods, among which are those that detect initial DNA damage in organisms of aquatic biocenosis. The microgel electrophoresis (MGE) "comet assay" was applied to a ubiquitous unicellular green alga (Chlamydomonas reinhardtii) to detect DNA damage caused by genotoxins. For this, the test protocol described by Singh NP et al. [Exp Cell Res 175: 184-191, 1988] was modified. Major modifications were the use of alkaline lysis buffer with ionic detergents and the reduction of preincubation and electrophoresis times. Short-time exposure of Chlamydomonas to the well-known genotoxicants 4-nitroquinoline-1-oxide (4-NQO), N-nitrosodimethylamine, and hydrogen peroxide led to dose-dependent DNA damage. Chlamydomonas responded very sensitively to treatment with increasing doses of 4-NQO. At a concentration of 25 nM, significant DNA damage was observed. At higher 4-NQO doses (> 100 nM), DNA damage was visible as complete DNA fragmentation into fine granules. N-Nitrosodimethylamine caused genotoxic effects at a concentration range from 0.014 to 0.14 mM without producing complete DNA fragmentation at the concentrations tested (highest dose, 140 mM). To evaluate the influence of illumination conditions during exposure, cells were incubated with increasing doses of H2O2 (0.25-1.0 mM) in darkness and in light. Our results indicate that incubation in light enables Chlamydomonas to cope with oxidative stress more efficiently than under dark conditions. To a certain extent, cytotoxic as well as genotoxic effects of H2O2 depend on the illumination condition or repair and anti-oxidative protection mechanisms activated by light, respectively.

[Colony blot method for detection of legionellas--results of a comparative study]. / Colony Blot-Methode zum Nachweis von Legionellen--Ergebnisse einer Vergleichsuntersuchung.

In the following short communication a new commercially available immunoassay for the quantitative detection of Legionellae after cultivation was compared with the conventional method recommended by ISO in a study shared by 6 laboratories. After a training phase of the laboratory personal a very good agreement of immunological and conventional method was observed by testing 310 water samples. The colony blot assay for quantitative identification of Legionella spec. is a rapid and specific method and can be recommended for quantification of Legionella spec. in water samples.

[Experiences with optimized methods for the detection of legionellae in drinking water]. / Erfahrungen mit optimierten Methoden zum Nachweis von Legionellen im Trinkwasser.

Entry of Legionellae into domestic water systems by passing through the drinking water distribution network has been assumed. To prove this question, samples were collected within a two years period at warm and cold water taps of households, the pipeline network and three water works of the city of Mainz, and examined for the presence of Legionellae. To detect even very small numbers of Legionellae, improvement of the conventional isolation procedure was necessary. Additionally, large volumes of cold water samples (50-250 L) were processed by using a pressure filter. For identification of Legionellae, an immunological rapid test (colony-blot-assay, own development) and a commercial gene-probe test (EnvironAmp Legionella Kits, Applied Biosystems) were enclosed in the programme. With the refined techniques the detection limit was improved while expenses of time and labour were reduced. Finally, the advantages and disadvantages of the single methods are discussed. For routine detection of Legionellae in the microbiological laboratory, a combination of effective methods is proposed which are easy to perform.

Rapid determination of members of the family Enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen.

An immunological method for the detection of members of the family Enterobacteriaceae in drinking water was developed. The method was based on a sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody immunoglobulin G2a 898 against enterobacterial common antigen. The enterobacterial common antigen sandwich ELISA combined with selective preenrichment culture could be performed in only 24 h. Six hundred sixty-eight water samples from a variety of German public water supplies were screened to verify the effectiveness of the new method. Ninety-eight percent of the results obtained by the immunological method could be confirmed by conventional microbiological methods. The immunological method proved to be considerably faster and more specific and sensitive than the standard method specified by the German drinking water regulations.

[Indoor acoustical requirements in school gymnasiums]. / Raumakustische Bedingungen in Schulsporthallen.

The acoustic quality of a gymnasium essentially determines the noise level which originates with use. High noise levels impair conditions of instructions and training. The time of reverberation was measured in 10 gymnasiums. The acoustic conditions were bad in most of them. It is not possible that more than one group of kids or students are under training together. Present sound absorbers on the ceiling do not meet all requirements.