Distribution of specific tetracycline and erythromycin resistance genes in environmental samples assessed by macroarray detection

A macroarray system was developed to screen environmental samples for the presence of specific tetracycline (Tc(R)) and erythromycin (erm(R)) resistance genes. The macroarray was loaded with polymerase chain reaction (PCR) amplicons of 23 Tc(R) genes and 10 erm(R) genes. Total bacterial genomic DNA was extracted from soil and animal faecal samples collected from different European countries. Macroarray hybridization was performed under stringent conditions and the results were analysed by fluorescence scanning. Pig herds in Norway, reared without antibiotic use, had a significantly lower incidence of antibiotic resistant bacteria than those reared in other European countries, and organic herds contained lower numbers of resistant bacteria than intensively farmed animals. The relative proportions of the different genes were constant across the different countries. Ribosome protection type Tc(R) genes were the most common resistance genes in animal faecal samples, with the tet(W) gene the most abundant, followed by tet(O) and tet(Q). Different resistance genes were present in soil samples, where erm(V) and erm(E) were the most prevalent followed by the efflux type Tc(R) genes. The macroarray proved a powerful tool to screen DNA extracted from environmental samples to identify the most abundant Tc(R) and erm(R) genes within those tested, avoiding the need for culturing and biased PCR amplification steps.     

A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.     

Biosynthesis of bile acids in a variety of marine bacterial taxa

Several marine bacterial strains, which were isolated from seawater off the island Dokdo, Korea, were screened to find new bioactive compounds such as antibiotics. Among them, Donghaeana dokdonensis strain DSW-6 was found to produce antibacterial agents, and the agents were then purified and analyzed by LC-MS/MS and 1D- and 2D-NMR spectrometries. The bioactive compounds were successfully identified as cholic acid and glycine-conjugated glycocholic acid, the 7alpha-dehydroxylated derivatives (deoxycholic acid and glycodeoxycholic acid) of which were also detected in relatively small amounts. Other masine isolates, taxonomically different from DSW-6, were also able to produce the compounds in a quite different production ratio from DSW-6. As far as we are aware of, these bile acids are produced by specific members of the genus Streptomyces and Myroides, and thought to be general secondary metabolites produced by a variety of bacterial taxa that are widely distributed in the sea.     

Reversible inhibition of bacterial bioluminescence by long-chain fatty acids

Long-chain unsaturated fatty acids, as well as certain saturated fatty acids such as lauric acid, are inhibitors of the in vivo luminescence of wild-type strains of four species of luminous bacteria (Beneckea harveyi, Photobacterium phosphoerum, P. fischeri, andP. leiognathi) as well as the myristic acid-stimulated luminescence in the aldehyde dim mutant M17 ofB. harveyi. Based on studies with the system in vivo, the principal site of action of all the fatty acids appears to be the reductase activity that converts myristic acid to myristyl aldehyde. This was confirmed by in vitro studies: Reductase activity in crude cell-free extracts is strongly inhibited by oleic acid.     

Linking metabolite production to taxonomic identity in environmental samples by (MA)LDI-FISH

One of the greatest challenges in microbial ecology remains to link the metabolic activity of individual cells to their taxonomic identity and localization within environmental samples. Here we combined mass-spectrometric imaging (MSI) through (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) with fluorescence in situ hybridization (FISH) to monitor antibiotic production in the defensive symbiosis between beewolf wasps and ‘Streptomyces philanthi'' bacteria. Our results reveal similar distributions of the different symbiont-produced antibiotics across the surface of beewolf cocoons, which colocalize with the producing cell populations. Whereas FISH achieves single-cell resolution, MSI is currently limited to a step size of 20–50 μm in the combined approach because of the destructive effects of high laser intensities that are associated with tighter laser beam focus at higher lateral resolution. However, on the basis of the applicability of (MA)LDI-MSI to a broad range of small molecules, its combination with FISH provides a powerful tool for studying microbial interactions in situ, and further modifications of this technique could allow for linking metabolic profiling to gene expression.Ecological analyses of microbial metabolites have thus far been hampered by the difficulty of localizing and quantifying these compounds in situ and tying their production to subpopulations or even single cells of individual microbial taxa. However, recent advances in mass-spectrometric imaging (MSI) techniques provide excellent tools to monitor metabolic processes and chemical communication in an ecological context (Svatoš, 2010, 2011). For example, matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) has successfully been employed to observe antagonistic interactions between Streptomyces and Bacillus strains in vitro (Yang et al., 2009). The analysis of microbial interactions in situ, however, requires the combination of metabolic profiling with taxonomic identification and localization of the involved microorganisms. Previous studies employing microautoradiography or high-resolution secondary ion mass spectrometry in combination with in situ hybridization have provided insights into the metabolism of individually identified bacterial cells in environmental samples (Orphan et al., 2001; Kindaichi et al., 2004; Behrens et al., 2008; Musat et al., 2008). However, the need for isotopic labeling limits the application of these techniques to a subset of biological questions.Here we combine MSI with fluorescence in situ hybridization (FISH) for simultaneous metabolite profiling and taxonomic identification of bacteria, using the defensive symbiosis between beewolf wasps and Streptomyces bacteria as a model. Beewolves of the genera Philanthus, Trachypus and Philanthinus (Hymenoptera, Crabronidae) cultivate ‘Streptomyces philanthi'' in specialized antennal gland reservoirs (Kaltenpoth et al., 2006; Goettler et al., 2007; Kaltenpoth et al., 2014) and secrete the bacteria into their subterranean brood cells before oviposition (Kaltenpoth et al., 2010a). Later, the larva incorporates the symbionts into the cocoon silk, where the streptomycetes produce a cocktail of at least nine different antibiotics (Kroiss et al., 2010) and thereby protect the larva against pathogenic fungi and bacteria during the long (up to 9 months) and vulnerable phase of hibernation (Kaltenpoth et al., 2005; Koehler et al., 2013). Previous studies using MSI revealed that the antibiotics abound on the outer surface of the cocoon, while they are virtually absent from the inner surface (Kroiss et al., 2010).We used (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) to visualize the abundance of two different antibiotics (piericidin A1 and B1) and subsequently localized the symbionts producing these compounds on beewolf cocoons using FISH. Pieces of beewolf cocoons were fixed to MALDI target plates without any pre-treatment using double-sided adhesive tape, with the outer cocoon surface facing upward. In order to allow for later alignment of ion-intensity maps and FISH images, the cocoon pieces were surrounded by thin paint markings (Edding751, 1–2 mm tip width, white), applied with the tip of a needle. This marker was chosen because it yielded characteristic signals in (MA)LDI-MS (measured at m/z 322.5±0.5) and also showed fluorescence at 640 nm, the excitation wavelength of the fluorescent dye used for FISH (that is, Cy5). MSI was carried out without any pretreatment of the samples (Hoelscher et al., 2009; Kroiss et al., 2010), or after application of 2,5-dihydroxybenzoic acid matrix by sublimation (Svatoš and Mock, 2013). A MALDI micro MX mass spectrometer (Waters, Milford, MA, USA) equipped with a nitrogen laser (337 nm) was used in the reflectron mode and positive polarity for data acquisition as previously reported (Kroiss et al., 2010). The step size in both x and y directions was set to 50 μm corresponding to 508 dots per inch resolution. Two-dimensional ion-intensity maps were reconstructed using the spectral data for the respective potassium adduct ions of piericidin A1 (PA1, m/z 454±0.5 [M+K]⁺) and piericidin B1 (PB1, m/z 468±0.5 [M+K]⁺) with the BioMAP software (Novartis Institutes for BioMedical Research, Basel, Switzerland). After (MA)LDI imaging, samples were subjected to FISH with the ‘S. philanthi''-specific probe SPT177-Cy5 (Kaltenpoth et al., 2005, 2006) as described previously (Kaltenpoth et al., 2010b). Fluorescence images were recorded on a Zeiss AxioImager Z.1 (Zeiss, Jena, Germany) using both the mosaic and z-stack options for obtaining high-resolution images with increased focusing depth. Overlays of (MA)LDI and FISH images were achieved in Adobe Photoshop CS5 Extended 12.0 by using the pen markings as a guide.(MA)LDI-MSI revealed a patchy distribution of antibiotics across the outer cocoon surface of European beewolves. The two measured antibiotic substances showed very similar distributions (Figures 1a–j), suggesting that both compounds—as well as possibly the other seven antibiotics produced by the symbionts on the beewolf cocoon that could not be measured here because of their low concentrations—are produced by individual bacterial cells or subpopulations of cells. This is supported by MSI with a high-resolution atmospheric pressure scanning microprobe (AP-SMALDI-MSI) of PA1 and PB1 produced by ‘S. philanthi'' on beewolf cocoons and in vitro, which confirmed the colocalization of both antibiotics (Figures 1k–n and Supplementary Figure S1, for experimental procedures see Supplementary Online Material). Thus, different symbiont subpopulations apparently do not specialize in the production of individual compounds, but instead produce a mixture of antibiotics.Open in a separate windowFigure 1(MA)LDI-FISH of antibiotics produced by symbiotic ‘Streptomyces philanthi'' bacteria on a beewolf cocoon (Philanthus triangulum) and in vitro. Ion-intensity maps of (a) the paint marker for alignment of LDI and FISH pictures (m/z 322.5); inset: image of the cocoon piece surrounded by white paint markings on the LDI target plate, (b) piericidin A1 (PA1, m/z 454.5 [M+K]⁺) and (c) piericidin B1 (PB1, m/z 468.5 [M+K]⁺). (d–f) The same maps, overlayed with a FISH micrograph of the cocoon piece. Symbiont cells were labeled with the fluorescent oligonucleotide probe SPT177-Cy5. (g–h) Magnifications of (e, f), respectively, with individual bacterial cells visible. (i–j) MALDI-FISH of (i) PA1 (m/z 454.5 [M+K]⁺) and (j) PB1 (m/z 468.5 [M+K]⁺) on another cocoon piece. (k–n) AP-SMALDI imaging of antibiotics produced by ‘S. philanthi'' in vitro. (k) Light microscopic image of an ‘S. philanthi'' colony, (l) PA1 (m/z 416.27 [M+H]⁺), (m) PB1 (m/z 430.25 [M+H]⁺), (n) overlay of PA1 (green) and PB1 (red).On the cocoon, FISH allowed for the visualization of individual symbiont cells, which were abundant across the entire cocoon surface and often occurred in highest densities along the outer cocoon threads (Supplementary Figure S2). The presence of the matrix had no influence on the efficiency of FISH after MSI (data not shown). The alignment of ion-intensity maps with FISH images revealed high concentrations of antibiotics around some subpopulations of cells, whereas other cell aggregations were surrounded by much lower amounts of antibiotics (Figures 1g–j), highlighting the possibility for cheating in the symbiosis. However, the current limitations in sensitivity and lateral resolution of MALDI-MSI do not permit the visualization of compounds on the single-cell level (~1 μm) and thereby may obscure fine-scale patterns of antibiotic production. This is supported by AP-SMALDI-MSI of beewolf cocoons at two different resolutions (step sizes 20 and 5 μm, Supplementary Figure S1): Whereas the high-resolution measurement revealed high concentrations of antibiotics along the outer cocoon threads, which agrees with the FISH experiments showing a similar pattern of symbiont cell densities (Supplementary Figure S2), this pattern was not as apparent at lower resolutions (Supplementary Figure S1). However, the high laser intensities required for a step size of 5 μm were destructive for the samples; therefore, subsequent FISH experiments could not be performed.The combination of (MA)LDI imaging and FISH provides a powerful tool for tying metabolite profiling to taxonomic identification in environmental samples. However, the laser intensity needs to be carefully adjusted for the desired application to achieve maximum sensitivity and resolution while at the same time conserving the structure of the sample. This problem can be circumvented by using desorption electrospray ionization (DESI) imaging, which we also successfully combined with FISH in preliminary experiments (data not shown). Still, the limitations of both (MA)LDI and DESI in lateral resolution and sensitivity currently prohibit single-cell resolution of metabolic profiling and restrict the technique to mapping the distribution of metabolites on the subpopulation level (Svatoš, 2011). Thus, the exploration of complex environmental samples is at present limited to microbial communities with distinct spatial structure. However, the major strength of MSI-FISH is its broad applicability to a wide range of small molecules as well as proteins (Svatoš, 2010). Therefore, (MA)LDI-FISH and DESI-FISH allow for addressing a multitude of questions in microbial ecology, ranging from interactions in mixed-species biofilms or cross-feeding associations to the chemical basis and dynamics of mutualistic and antagonistic encounters. As several signal enhancement techniques for FISH have been developed (for example, Schönhuber et al., 1997; Zwirglmaier, 2005), modifications of the approach described here could also be employed to tie metabolic profiling by (MA)LDI or DESI imaging to the presence (Moraru et al., 2010) or expression (Pernthaler and Amann, 2004) of particular genes of interest in microbial communities or eukaryotic tissues. Future studies should explore the possibility for using oligonucleotide labels and hybridization protocols that allow for simultaneous MSI of labeled cells and metabolites of interest, which would obviate the necessity for subsequent FISH and thereby circumvent the problems with high laser intensities. Alternatively, new MALDI matrices capable of dissipating the high ultraviolet-laser intensities and thus preventing DNA damage could be developed.

Table 1

Comparison of established methods for linking localization and taxonomic identification of microbes to the production of particular metabolites of interest in environmental samples

Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples

Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymographic procedure that specifically detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA fluorescent probe, which is copolymerized in sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis and renaturation, enzymatic digestions of the substrate in 50 mM of Tris buffer at pH 7.4 generates fluorescence emission at 695 nm. The enzymatic activities can be analyzed directly by conventional gel imaging systems with a detection limit of 40 pg. This protocol is fast (hours) and does not require staining and destaining steps. The procedure is independent of plasminogen and, therefore, can efficiently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demonstrated a highly correlative relationship (r2=0.999) between the amount of uPA (over the range of 0.1-8.0 ng) and the average intensity of the fluorescent band. We were able to directly measure uPA activities in different cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.     

Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8

Quantitative PCR is becoming the method of choice for the detection of pathogenic microorganisms and other targets in the environment. A major obstacle when amplifying DNA is the presence of inhibiting substances like humic acids that decrease the efficiency of PCR. We combined the polymeric adsorbent Supelite™ DAX-8 with a large-volume (10 mL) nucleic acid extraction method to decrease the humic acid content prior to qPCR quantification in water samples. The method was tested by spiking with humic acid standards and the bacterial surrogate Acinetobacter baylyi ADP1. Improvements in qPCR detection of ADP1 after application of DAX-8 resin (5 and 10 w/v%) were compared with the effects of added bovine serum albumin (BSA) (50, 100 and 200 ng/μL). Both additions improved detection of ADP1 by counteracting inhibitory effects. There were no changes in mean cycle threshold difference (ΔC_T) after application of DAX-8 compared to the control despite some loss of DNA, whereas significant increases occurred for BSA, irrespective of BSA concentration applied. The use of DAX-8 leads to an increase in qPCR amplification efficiency in contrast to BSA. The commonly used method to calculate genomic sample concentrations by comparing measured CT values relative to standard curves is only valid if amplification efficiencies of both are sufficiently similar. DAX-8 can provide this efficiency by removing humic acids permanently from nucleic acid extracts and has the potential to significantly increase the reliability of reported non-detects and measured results obtained by qPCR in environmental monitoring.     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.     

Metabolism of saturated 1-14C-labelled fatty acids in the silkworm Bombyx mori L

1. Only a small percentage of 1-(14)C-labelled saturated fatty acids injected in the silkworm is respired as carbon dioxide. 2. The rate of utilization of fatty acids is low both at the larval and pupal stages. 3. The insect has the ability to elongate C(12) and C(16) saturated fatty acids and to desaturate C(18) saturated fatty acids. 4. Much of the administered radioactivity is found in the triglyceride fraction, followed by the phospholipid and diglyceride fractions. 5. Diglycerides seem to be the transport form of fatty acids. 6. The insect seems to metabolize both natural and unnatural fatty acids in the same manner.     

Synthesis of cyclopropane fatty acids in isolated bacterial membranes

Isolated E. coli membrane vesicles can synthesize cyclopropane fatty acids from S-adenosylmethionine using endogenous membrane phospholipid as the lipidsubstrate. The major methylated products are methylenehexadecanoic acid and methyleneoctadecenoic acid esterified to phosphatidylethanolamine. The membrane system is slightly stimulated by sodium dodecyl sulfate at low concentrations but is inhibited by neutral surfactants. The reaction is inhibited by phospholipase C and phospholipase A. The Arrhenius plot for the enzyme reaction is discontinuous over the temperature range 0–35 °C.     

Selected ion monitoring in quantitative gas-liquid chromatographic-mass spectrometric detection of fatty acid methyl esters from environmental samples

To calculate selected ion monitoring (SIM) gas-liquid chromatography (GLC)-mass spectrometry (MS) results of phospholipid fatty acids (PLFAs) from environmental samples, coefficients were calculated for each fatty acid by dividing the sum of ion intensities in SCAN with that of ions followed in SIM. The SIM chromatogram areas were multiplied with the coefficients, and then processed as in SCAN. The results were compared to those obtained using calibration curves and SCAN. The calibration curve and coefficient based results had the greatest errors of 7.8 and 6.7%, respectively, outside standard deviations of SCAN percentages. The PLFA contents calculated using calibration curves and coefficients were 104.9+/-7.3% and 101.5+/-8.6%, respectively, of SCAN values. SIM increased sensitivity approximately 10-fold from SCAN, and the smallest detectable injected amount was approximately 50 ng (0.18 nmol) for 20 fatty acids, corresponding to 4 x 10(6) cells.     

Inhibition of chymase activity by long chain fatty acids

The activity of chymase was markedly inhibited by fatty acids with carbon chain lengths of 14-22 at doses greater than 0.02 microM, irrespective of the number of double bonds. Cis acids with a carbon chain length of 18, such as stearic acid, oleic acid, linoleic acid, and linolenic acid were potent inhibitors, whereas the trans isomer of oleic acid, elaidic acid, showed less inhibitory activity. The extent of inhibition by oleyl alcohol was almost the same as that by oleic acid, suggesting that the acid moiety itself was not necessary for the inhibition; but a fatty acid with a terminal functional amide, oleamide, showed little inhibitory activity. The inhibition was noncompetitive and was reversible, and the Ki value of oleic acid was 2.7 microM. Stearic acid and oleic acid inhibited all chymotrypsin-type serine endopeptidases tested. The ID50 values of these fatty acids for atypical mast cell protease were higher than those for the other chymotrypsin-type serine endopeptidases tested. Other proteases, such as papain, trypsin, collagenase, and carboxypeptidase A, except cathespin D, were not affected by stearic or oleic acid.     

Detection of bacterial pathogens in environmental samples using DNA microarrays

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.     

Aromatic amino acids in amniotic fluid samples analyzed by reversed-phase liquid chromatography with spectrophotometric detection

The applicability of threshold logic units, a form of nonparametric pattern recognition, to the processing of metabolic profile data obtained by high-efficiency glass capillary column gas chromatography has been investigated. The test data included profiles of the volatile constituents of urine from normal individuals and from individuals with diabetes mellitus. A feature extraction algorithm allowed for dimensionality reduction and indicated the constituents most important in the normal versus pathological distinction. With an optimum number of dimensions, a normal versus pathological prediction rate of 93.75% was achieved. Gas chromatography—mass spectrometry was utilized to identify important profile constituents.     

Titration and exchange studies of liver fatty acid-binding protein with 13C-labeled long-chain fatty acids

Uniformly (13)C-labeled long-chain fatty acids were used to probe ligand binding to rat liver fatty acid-binding protein (LFABP), an atypical member of the fatty acid-binding protein (FABP) family that binds more than one molecule of long-chain fatty acid, accommodates a variety of diverse ligands, and exhibits diffusion-mediated lipid transport to membranes. Two sets of (1)H-(13)C resonances were found in a titration series of NMR spectra for oleate-LFABP complexes, indicating that two molecules of the fatty acid are situated in the protein cavity. However, no distinct resonances were observed for the excess fatty acid in solution, suggesting that at least one ligand undergoes rapid exchange with oleate in the bulk solution. An exchange rate of 54 +/- 6 s(-1) between the two sets of resonances was measured directly using (13)C z,z-exchange spectroscopy. In light of these NMR measurements, possible molecular mechanisms for the ligand-exchange process are evaluated and implications for the anomalous fatty acid transport mechanism of LFABP are discussed.     

Development of 18S rRNA-targeted oligonucleotide probes for specific detection of Hartmannella and Naegleria in Legionella-positive environmental samples

Aquatic protozoa are natural hosts of the human pathogen Legionella pneumophila. The fluorescence labeled 16S rRNA-targeted oligonucleotide probe LEGPNE1 has recently been shown to specifically detect extracellular legionellae as well as intracellular legionellae parasitizing protozoa. In this study we designed oligonucleotide probes which are complementary to distinct regions of the 18S rRNA of the Legionella host organisms of the genera Hartmannella and Naegleria. The specificity of the probes, HART498 and NAEG1088, was tested by in situ hybridization of various laboratory reference strains. In order to evaluate the fluorescent probes for environmental studies three selected Legionella-positive cold water habitats were examined for the presence of these protozoa. Traditional culture methods followed by morphological identification revealed an almost consistent presence of Naegleria spp. in cold water habitats. Other protozoa species including Acanthamoeba spp., Echinamoeba spp., Hartmannella spp., Platyamoeba placida, Saccamoeba spp., Thecamoeba quadrilineata, and Vexillifera spp. were found sporadically. Concomitant analysis of the pH, conductivity and temperature of the water samples revealed no preference of Legionella or the respective protozoa for certain environmental conditions. The specificity of the newly designed 18S rRNA probes demonstrates that they are valuable and rapid tools for the identification of culturable environmental protozoa.