Quorum sensing: bacterial chatting.
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Adaptation, Physiological↗
Biomedical subjects
Ursula Obst
Publications and source records attributed to Ursula Obst.
Use of quantitative real-time RT-PCR to analyse the expression of some quorum-sensing regulated genes in Pseudomonas aeruginosa.
P. aeruginosa living in biofilm populations sends out diffusive signalling molecules, called autoinducers, for example acylated homoserine lactone (AHL) or the P. aeruginosa quinolone signal (PQS). So far, two quorum-sensing systems, LasR and VsmR, have been identified in P. aeruginosa, both of which are required for all virulence determinants. The expression of specific genes involved in quorum-sensing regulatory mechanisms has been analysed with molecular biology methods. Real-time quantitative PCR is a highly sensitive and powerful technique for quantification of nucleic acids. Expression of the genes vsmR, lasI, and PA4296 was studied by use of reverse transcriptase and subsequent quantitative real-time PCR of the cDNAs. In parallel, expression of ribosomal 16S rRNA, used as a housekeeping gene that was constitutively expressed in all analyses, was also monitored. Biofilm was compared with planktonic bacteria, and in contrast to vsmR and Pa4296, the lasI gene was found to be down-regulated in biofilm. Extended experiments were run with synthetic signal molecules inducing regulated processes in bacterial populations. It was shown that the genes under investigation were up-regulated in mature biofilm in the presence of the signal molecule N-(3-oxododecanoyl)-L-homoserine lactone.
4-Butyrolactone↗
UV-induced dark repair mechanisms in bacteria associated with drinking water.
Caulobacter crescentus and Aquabacterium commune, both isolated from drinking water, as well as environmental isolates of Pseudomonas aeruginosa and Enterococcus faecium were treated with different UV fluences to study their capacity to restore induced DNA damages. Here, the induction of a key mechanism of bacterial dark repair, the so-called recA system, was analysed. With newly designed probes, the specific recA mRNA was detected by Northern blot. Additionally, the RecA protein was measured by the Western blot technique using a specific antibody. In drinking water bacteria as well as in opportunistic microorganisms, a specific induction of dark repair mechanisms was found even at UV fluences higher than 400J/m(2), the German standard for UV disinfection. This induction depended on the incubation time after UV treatment. Nevertheless, the UV-induced recA expressions were found to differ in the bacteria under investigation.
Bacteria↗
Evaluation of inhibition and cross-reaction effects on real-time PCR applied to the total DNA of wastewater samples for the quantification of bacterial antibiotic resistance genes and taxon-specific targets.
In order to evaluate the applicability of six primer and probe sets for TaqMan real-time RCR on DNA of wastewater samples, effects of the sample matrix and DNA background on target quantification were studied with respect to differences between functional genes and taxonomically used rDNA targets. Primer/probe assays for real-time PCR (TaqMan) designed to quantitatively detect the antibiotic resistance genes bla(VIM), vanA, ampC, mecA, and taxon-specific 23S rDNA sequences for Pseudomonas aeruginosa and Enterococcus faecium/faecalis were tested for their sensitivity and amplification robustness. The amplification of their gene targets in DNA extracts of wastewater ("wastewater DNA") and reference strains ("reference DNA") was compared with their amplification in "model DNA" which was composed of wastewater DNA and reference DNA. Target detection was quantifiable along up to seven decimal orders of magnitude. For the detection of the resistance genes bla(VIM), vanA, ampC, and mecA as well as the enterococci directed PCR only weak or no inhibition due to the impurities or wastewater DNA matrix were demonstrated for the applied target concentrations. The taxonomically applied detection system for the quantification of P. aeruginosa showed a limited performance. For the analysis of the amplification dynamics of possibly similar nucleotide sequences of organisms related to P. aeruginosa a SYBR Green assay was employed. Competitive amplification of similar sequences was identified to be a major mechanism of reduced sensitivity. Hence, the primers were modified for an optimised detection. With the resulting reduction of cross reactions an increased sensitivity was achieved for the detection and quantification of P. aeruginosa in wastewater DNA.
Amino Acid Sequence↗
The quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) enhances the host defence by activating human polymorphonuclear neutrophils (PMN).
The P. aeruginosa quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) interacts not only with bacteria, but also with mammalian cells, among others with those of the immune defence system. We focussed on the possible interaction of 3OC12-HSL with human polymorphonuclear neutrophils (PMN), because these cells are the first to enter an infected site. We found that 3OC12-HSL attracts PMN, and up-regulates expression of receptors known to be involved in host defence, including the adhesion proteins CD11b/CD18 and the immunoglobulin receptors CD16 and CD64. Furthermore, the uptake of bacteria (phagocytosis), which is crucial for an efficient defence against infection, was enhanced. Thus, recognising and responding to 3OC12-HSL not only attracts the PMN to the site of a developing biofilm, but also reinforces their defence mechanisms, and hence could be a means to control the infection in an early stage and to prevent biofilm formation.
4-Butyrolactone↗
Real-time PCR detection of Pseudomonas aeruginosa in clinical and municipal wastewater and genotyping of the ciprofloxacin-resistant isolates.
Real-time quantification of Pseudomonas aeruginosa was performed in various wastewater systems including clinical, municipal wastewaters and inflow from a wastewater treatment plant. The highest concentrations of P. aeruginosa-specific targets were detected in clinical wastewaters. Limitations of the detection system resulting from inhibition or cross-reaction were identified. Ciprofloxacin-resistant P. aeruginosa strains were isolated after specific enrichment from clinical and municipal wastewaters. In some cases they were also cultivated from effluent of a wastewater treatment plant, and from its downstream river water. A total of 119 isolates were phenotypically characterized as ciprofloxacin-resistant via antibiogram testing. Subsequently, the fluoroquinolone-resistance-mediating mutations in the genes gyrA codon positions 83 and 87, gyrB codon position 466 and parC codon positions 87 and 91 were determined by mini-sequencing. Ciprofloxacin resistance was mainly associated with mutations in gyrA codon position 83 and parC mutation in codon positions 87 or 91 of the bacterial gyrase and topoisomerase II genes. All ciprofloxacin-resistant P. aeruginosa strains were compared with genotypes from clinical data of fluoroquinolone-resistant P. aeruginosa infections. The results were in agreement with data from clinical analyses, with the exception that no gyrA 87 and no gyrB mutations were found in ciprofloxacin-resistant P. aeruginosa wastewater isolates.
Ciprofloxacin↗
Induction of neutrophil chemotaxis by the quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone.
Acyl homoserine lactones are synthesized by Pseudomonas aeruginosa as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.
4-Butyrolactone↗
Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan).
Real-time PCR assays were developed for the quantifiable detection of the antibiotic-resistance genes vanA of enterococci, ampC of Enterobacteriaceae, and mecA of staphylococci in different municipal wastewater samples. Primer and probe designs for these resistance genes were constructed and optimised for application in standardised TaqMan PCR assays. Using reference strains, the linear measurement ranges of the assays were defined and covered concentration ranges of five to seven exponential values. Wastewater isolates of vancomycin-resistant enterococci (VRE) and beta-lactam-resistant Enterobacteriaceae were cultivated from municipal wastewaters in order to verify the specificity and sensitivity of the primer-probe systems. Additionally, clinical strains of staphylococci resistant to methicillin (MRSA) confirmed the applicability of the mecA-specific detection system. Total DNAs were extracted from five different wastewater treatment plants and used for direct TaqMan PCR detection of the resistance genes without prior cultivation. In municipal wastewater, the resistance gene vanA was detected in 21% of the samples, and ampC in 78%. The gene mecA was not found in municipal wastewater, but in two clinical wastewater samples.
Bacterial Proteins↗
Investigation of natural biofilms formed during the production of drinking water from surface water embankment filtration.
Populations of bacteria in biofilms from different sites of a drinking water production system were analysed. Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analyses revealed changing DNA band patterns, suggesting a population shift during bank filtration and processing at the waterworks. In addition, common DNA bands that were attributed to ubiquitous bacteria were found. Biofilms even developed directly after UV disinfection (1-2m distance). Their DNA band patterns only partly agreed with those of the biofilms from the downstream distribution system. Opportunistic pathogenic bacteria in biofilms were analysed using PCR and Southern blot hybridisation (SBH). Surface water appeared to have a direct influence on the composition of biofilms in the drinking water distribution system. In spite of preceding filtration and UV disinfection, opportunistic pathogens such as atypical mycobacteria and Legionella spp. were found in biofilms of drinking water, and Pseudomonas aeruginosa was detected sporadically. Enterococci were not found in any biofilm. Bacterial cell counts in the biofilms from surface water to drinking water dropped significantly, and esterase and alanine-aminopeptidase activity decreased. beta-glucosidase activity was not found in the biofilms. Contrary to the results for planktonic bacteria, inhibitory effects were not observed in biofilms. This suggested an increased tolerance of biofilm bacteria against toxic compounds.
Anti-Bacterial Agents↗
Approaches to bioresponse-linked instrumental analysis in water analysis.
A new concept based on hyphenation of biotests, for biological selection, and chemical analysis is introduced for water analysis. Biomolecular recognition components such as receptors, enzymes, and nucleic acids integrated in biological reaction chains are used for binding and selective enrichment of known and unknown biologically active substances in water samples; this is followed by identification and quantitation. The coupling of biomolecular recognition and binding to chemical analysis can be achieved either in discrete analytical steps, e.g. binding and elution of bioactive ligands from affinity columns followed by chemical analysis, or by methods capable of monitoring the binding of the ligand and simultaneous verification of its identity. This analytical strategy, denoted bioresponse-linked instrumental analysis (BLIA), enables detection of potential biological effects and identification of the analyte causing these effects. Several examples are presented.
Biosensing Techniques↗
Analysis of non-covalent protein complexes by capillary electrophoresis--time-of-flight mass spectrometry.
A capillary electrophoresis-electrospray ionisation time-of-flight mass spectrometry (CE-ESI-TOF-MS) method for characterisation of non-covalent protein complexes is described using a coaxial liquid sheath-flow sprayer. The CE capillary was connected to the mass spectrometer using a commercial CE-MS sprayer mounted on a ceramic holder of the ESI interface of the mass spectrometer. Using myoglobin (Mb) as an example of non-covalent protein complex, the effect on complex stability caused by organic modifiers added to the sheath liquid was analysed. Depending on the amount of methanol, either intact Mb or the apoprotein and the prosthetic heme group were detected.
Electrophoresis, Capillary↗
Congruence in the performance of model nitrifying activated sludge plants located in Germany, Scotland and Spain.
A laboratory model nitrifying activated sludge plant treating OECD synthetic sewage was designed and constructed by each of three laboratories in Germany, Scotland and Spain in order to produce a sludge inoculum for 5 rapid toxicity bioassays. The plants were run for 3 years and produced sludge for the microbially based bioassays Vibrio fischeri bioluminescence, ATP luminescence and respiration, and, nitrification and enzyme inhibition. Although the initial sludge inoculum for the plants differed, as did some of the running conditions such as temperature regime, the sludge produced within the different countries had similar characteristics with respect to sludge age, total suspended solids and volatile suspended solids. Nitrification was generally maintained over the 3-year period although there were occasions when the process was inconsistent. Nitrification recovery was afforded by reseeding with a nitrifying sludge from a local wastewater treatment works (WWTW) or imposition of starvation conditions for a period of time. The sludge produced was used to carry out toxicity testing and results compared well with those using sludge from a WWTW. Overall, the use of sludge generated in the laboratory could be used for toxicity testing negating the need to resort to the use of natural WWTW sludge, which may contain a range of toxic substances due to uncontrolled industrial and domestic inputs and an unbalanced microbial consortium.
Adenosine Triphosphate↗
Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples.
A recent PCR detection technique (TaqMan) based on the 5'-3'-exonuclease activity of the Taq DNA polymerase was applied to the detection of indicator organisms in water samples. In this technique, an increasing fluorescence signal is measured online which enables direct assessment of results after PCR without additional detection steps. The test is completed within about 5 h. Two sets of primers and probes were designed and tested: a genus-specific assay for the detection of Enterococcus spp. based on 23S rRNA sequence and an Escherichia coli-specific assay based on the uidA gene sequence. Specificity of the assays was confirmed by testing strains of target bacteria and potential interfering microorganisms. Application of the tests to 55 natural water samples showed the need of an overnight enrichment step to achieve compliance with detection limits of existing regulations. Compared with a parallel microbiological examination of the samples, agreement was 96% with the Enterococcus assay and 98% with the E. coli assay. The rapidity and feasibility of the method point to benefits in drinking water analysis, particularly in emergency situations and, thus, to improved public health management.
Enterococcus↗
Strategies of maintaining the natural purification potential of rivers and lakes.
Surface waters have a great impact on ecology and life of man. Because of their current use and exploitation, chemical and biological pollution, as well as physical changes of rivers and lakes are common. Natural self-purification potential helps to compensate harms to a certain degree. Proposals are given to detect restrictions of the purification potential in time as well as to support and to enhance the natural purification potential.
Biodegradation, Environmental↗