Combination of rRNA-Targeted Hybridization Probes and Immuno-Probes for the Identification of Bacteria by Flow Cytometry

System. App!. Microbio!. 19, 569-576 (1996) © Gustav Fischer Verlag

Combination of rRNA-Targeted Hybridization Probes and Immuno-Probes for the Identification of Bacteria by Flow Cytometry GUNTER WALLNER\ IVO STEINMETZ2, DIETER BITTER-SUERMANN2, and RUDOLF AMANN 3 GSF-Forschungszentrum fur Umwelt und Gesundheit, AG Ourchflugzytometrie, 0-85764 Oberschleigheim, Germany Medizinische Hochschule Hannover, Institut fur Medizinische Mikrobiologie, 0-30625 Hannover, Germany Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen, 0-80290 Miinchen, Germany

Summary Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were for the first time combined for the flow cytometric identification of bacteria. Artificial mixtures of fixed cells were hybridized with fluorescein-labeled, rRNA-targeted oligonucleotide probes and stained indirectly with biotinylated antibodies and R-phycoerythrin (PE) conjugated streptavidin. Finally, they were counterstained with 4',6diamidino-2-phenylindole (OAPI) in order to discriminate cells from background. Forward scatter, fluorescein- as well as PE- and OAPI-fluorescence were measured simultaneously for the differentiation of bacterial species by multiparameter flow cytometric analysis. The influence of detergents in the hybridization buffer on nonspecific antibody binding was evaluated. Sodium dodecyl sulfate (SOS) induced strong nonspecific staining and was, therefore, replaced by Tween 20. We found that the immunostaining steps can be performed before or after hybridization. This combination of rRNA-targeted hybridization probes and immuno-probes for flow cytometry makes possible the highly specific and automated identification of micro-organisms at any desired taxonomic leve!.

Key words: Flow Cytometry - Bacteria - Identification - Ribosomal RNA - Fluorescent in situ Hybridization - Immunofluorescence - Three Color Analysis

Introduction Classical methods for the detection and identification of micro-organisms require their cultivation in pure cultures. These methods are time- and labor-intensive, even if they are combined with automated identification systems. Often, only a minor fraction of the bacteria actually present in a natural sample can be grown under standard conditions. Due to peculiar growth requirements or to a state of nonculturability, only 0.001 % to 10% of the direct microscopic counts could be recovered by plate count or mostprobable-number techniques from a broad range of environmental samples (d. Amann et aI., 1995, and references therein). Moreover, the inevitable selectivity of cultivation techniques is hardly reflecting the true microbial community composition. Recent microscopic studies with rRNAbased probes have demonstrated this limitation even in activated sludge samples, from which a relatively high percentage of the bacterial cells (up to 15%) could be grown on agar plates (Wagner et al. 1993, Manz et al. 1994). In contrast, combined with appropriate staining techniques, direct flow cytometric or microscopic analyses of individual cells in fresh or fixed samples can reveal micro-

bial community structures and cellular properties as they were in situ at the time of sampling. The advantages of flow cytometry over microscopy are its high speed and automation. Several parameters based upon light scattering or fluorescence can be measured simultaneously for each cell at rates of more than 10 3 cells per second. For the flow cytometric identification of bacteria simple staining techniques with dyes like propidium iodide (staining DNA and RNA) and fluorescein isothiocyante (staining proteins) have been suggested (Miller and Quarles, 1990). However, the cellular constituents to which these stains bind are present in all cells and their contents depend more on growth conditions and growth phase than on species peculiarities. Therefore, such dyes, as well as narrow and wide angle light scattering (Allman et aI., 1993), can be used only for the identification of isolates which belong to a small set of bacterial species to choose from and which have to be cultured under highly standardized conditions. DNA base composition is a parameter that is constant for each species and that can be measured flow cytometrically by a combination of GC- and AT-

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G. Wallner, I. Steinmetz, D. Bitter-Suermann, and R. Amann

selective DNA-stains (Van Dilla et al., 1983; Sanders et al., 1990). But again, it can be exploited only for the differentiation of species belonging to a known set and differing sufficiently in their genomic %G+C content. Only reagents that bind to structures (or yield patterns) that are unique for a taxon allow an unambiguous identification. Fluorescently labeled antibodies and nucleic acid probes can meet this requirement and, consequently, have been applied to the flow cytometric identification of bacteria. Whereas antibodies require pure cultures of target organisms for their production and are usually species or strain specific, nucleic acid probes directed against rRNA can be constructed for as yet nonculturable organisms using PCR for retrieval of rRNA sequences (Amann et al., 1995, and references therein). Their specificity can be rather freely and deliberately chosen between the subspecies and the kingdom level (Stahl and Amann, 1991), depending on the degree of evolutionary conservation of the target region. In addition, rRNA-targeted probes can be used to assess general metabolic activity of individual cells, because rRNA contents and, consequently, intensities of the hybridization signals have been found to be directly correlated with growth rates of bacterial cultures (DeLong et al., 1989; Poulsen et al., 1993; Wallner et al., 1993). Immunofluorescence has previously been used for the flow cytometric detection and identification of bacteria in cultures (Ingram et al., 1982; Phillips and Martin, 1983; 1988), saliva (Sahar et al., 1983), cooling tower water (Tyndall et al., 1985), milk (Donnelly and Baigent, 1986), and activated sludge (Volsch et al., 1990) or of protozoa in water samples (Vesey et al., 1994). Up to now, flow cytometry and rRNA-targeted probes have mainly been applied to cultured bacterial, yeast, protist, or algal cells and mixtures thereof (Amann et al., 1990b; Zarda et al., 1991; Lim et al., 1993; Wallner et al., 1993; Simon et al., 1995), but recently their applicability to the identification and characterization of microbial cells in such a complex microbial community as activated sludge has been demonstrated (Wallner et al., 1995). In this study we describe the simultaneous use of rRNAtargeted probes, which are in general specific at the species level and above, and antibodies, which are frequently specific at the species level and below. The combination of these two highly specific molecular tools opens up the whole range of phylogenetic levels for the high resolution and automated identification of micro-organisms by flow cytometry.

Materials and Methods Organisms and culture conditions. Comamonas testosteroni (DSM 50244T; DSM = Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany), E. coli K12 C600 (Peters et al., 1985), as well as Pseudomonas aeruginosa strain ATCC 33351 (serotype 4; ATCC = American Type Culture Collection, Rockville, MD, USA) and strain 492 (produces alginate; culture collection of the Institut fur Medizinische Mikrobiologie, Medizinische Hochschule Hannover) were grown

aerobically in YT-broth (g 1-1: tryptone, 10; yeast extract, 5; glucose, 5; sodium chloride,S; pH 7.2). The cells were harvested during exponential growth, fixed with 3% paraformaldehyde (Amann et al., 1990b), and stored in 50% ethanoUphosphate buffered saline (PBS; 130 mM sodium chloride, 10 mM sodium phosphate buffer, pH 7.2). Oligonucleotide probes. The following oligodeoxynucleotides were used as hybridization probes: EUB338, complementary to a region of the 16S rRNA of all Bacteria (Amann et al., 1990a); GAM42a and BET42a, complementary to a region of the 23S rRNA conserved in the gamma- and beta-subclass of the Proteobacteria, respectively (Manz et al., 1992). The probes were synthesized with a C6-TFA aminolinker [6-(trifluoroacetylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite] at the 5'-end and labeled with FLUOS [5(6)-carboxyfluorescein-Nhydroxysuccinimide ester; Boehringer Mannheim, Mannheim, Germany] as described in Amann et al. (1990a). Immuno-probes. The monoclonal antibody (mAb) antiECA898 (immunoglobulin subclass G2a ) binds to enterobacterial common antigen (Peters et al., 1985), mAb anti-PAE2592 (IgG3 ) is specific for the lipopolysaccharide of P. aeruginosa serotype 4 (I. Steinmetz, unpublished results), and mAb anti-PAE2596 (lgG 2b ) is specific for P. aeruginosa alginate (I. Steinmetz, unpublished results). Antibodies were biotinylated with biotinamidocaproate N-hydroxy-succinimide ester (Sigma, Deisenhofen, Germany) as described by Harlow and Lane (1988) and adjusted to a concentration of 1 mg/ml. R-phycoerythrin conjugated streptavidin was obtained from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, USA). Optimal antibody and streptavidin concentrations were determined by dilution series. Staining. FISH: Approximately 106 cells were hybridized in a buffer containing 0.9 M NaCl, 0.05% Tween 20, 20 mM Tris/ HCI (pH 7.2), and 2 ng/!tl oligonucleotide probe at 46°C for 2 hours (Wallner et al., 1993), and subsequently washed in hybridization buffer without probe (46°C, 20 min). For probe GAM42a that requires more stringent hybridization conditions 35% formamide was added to the buffer. Equimolar unlabeled competitor BET42a was added to probe GAM42a for enhanced single mismatch discrimination (Wallner et al., 1995). Immunostaining: Cells were incubated in buffer A (PBS, pH 7.5, containing 1% bovine serum albumin [Merck, Darmstadt, Germany]) with biotinylated antibody (1: 30) at 22°C for 60 min, washed in buffer A, and stained with PE-conjugated streptavidin (1 : 50 in buffer A) at 22°C for 30 min. In order to keep cell loss by centrifugation low, cells were washed only once with buffer A after every staining step. Finally, the cells were resuspended in PBS (pH 8.4) containing 1 !tM DAPI (4',6-diamidino-2-phenylindole, dilactate; Polysciences, Warrington, PA, USA). All buffers were filtered through 0.2 !tm pore size for flow cytometric analysis. Flow cytometry. Flow cytometric analyses were performed on a FACStar Plus (BD, Becton Dickinson, San Jose, CA, USA) equipped with two argon ion lasers. The first laser was adjusted to the 488 nm line (500 mW) for the measurement of forward angie light scatter (488 nm band pass filter for detection, BD 488 DF) as well as of fluorescence of the hybridization probes (green fluorescein fluorescence: 530 nm band pass, BD 530 DF; photomultiplier voltage: 600 V) and immuno-probes (orange-red PE fluorescence, reflected by a BD 560 DM dichroic mirror: 575 nm band pass, BD 575 DF; 700V). The second laser (UV multilines 351.1-363.8 nm, 200 mW) was used to excite DAPI (blue DAPI fluorescence: 424 nm band pass, BD 424 DF; 700 V). The four parameters were acquired in list mode as pulse height signals (four decades in logarithmic scale). Data analysis. Flow cytometric data were analyzed using the DAS software package (Data Analysis System, DAS V4.2; Beis-

Identification of Bacteria by Flow Cytometry

ker, 1994). Gates for statistical calculations and graphic presentations were set in two-dimensional histograms. In Figures 2 and 3 each dot represents a cell, its position the respective values for the parameters on the x- and y-axis measured for this cell, given in arbitrary units. Frequencies can be estimated from the density of the dots and the overlayed contour lines (3, 6, 12,24,48, and 90% of maximum frequency, outer to inner lines). As the fluorescence emission spectra of fluorescein and PE are overlapping and optical filters cannot completely separate light emitted from these two f1uorochromes, this effect was compensated by computational calculations in order to enhance the discrimination of probe- and antibody-positive against negative cells. Using the DAS software package, the original (uncompensated) fluorescence values were transformed by substraction of the fraction of fluorescence from the unwanted fluorochrome, creating two additional parameters FLPE-comp and FLFL-comp according to the following equations:

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FLFL-comp = FLFL - kPEintoFL * FLpE FLPE-comp = FLpE - kFLinroPE -- FLFL where FL pE and FL FL are the values measured by the fluorescence detectors for PE and fluorescein, respectively; kPEintoFL and kFLinroPE are the fractions of fluorescence from PE in the fluorescein channel, and vice versa; FLPE-comp and FLFL-comp are the actual (compensated) fluorescence values emitted from PE and fluorescein, respectively. The compensation factors for the instrument settings described above were determined as kPEintoFL = 0.025 and kFLintoPE = 0.063 from measurements of mono-labeled cells (as in Fig.2A and B).

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Results and Discussion

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Nonspecific immunofluorescence caused by detergent In initial experiments strong nonspecific IF of negative control cells occurred when the immunostaining steps were performed subsequent to our standard hybridization procedure. The hybridization buffer in this protocol contained 0.1 % SDS as detergent for improving permeability of fixed bacterial cells to oligonucleotide probes (Wallner et a1., 1993). By varying the composition of the hybridization buffer (Fig. 1) it was found that SDS causes strong nonspecific antibody binding. The fluorescence of SDStreated nontarget cells was slightly increased after incubation with PE-streptavidin alone (Fig. 1D), indicating that SDS may also induce some nonspecific binding of the PE conjugated streptavidin. Most of the nonspecific signal, however, was due to nonspecific binding of the negative control antibody (Fig. IF), resulting in cellular fluorescence almost as high as after staining with a specific antibody (Fig. 1G). Triton X-100 also gave rise to considerable nonspecific staining (Fig. IE). In contrast, cells treated with buffers containing Tween 20 (Fig. 1C) or formamide (Fig. 1B) did not show nonspecific IF. The fluorescence measured for these cells in the PEchannel was as low as for unstained cells (essentially instrument background). Similar results were found for other combinations of E. coli or P. aeruginosa ATCC 33351 and strain 492 cells with antibodies anti-ECA898, antiPAE2592, and anti-PAE2596 (data not shown). Therefore, SDS in the hybridization buffer was replaced by Tween 20 for subsequent experiments. Formamide could be added in order to increase stringency of the hybridization conditions.

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Fig. 1. Induction of nonspecific immunofluorescence by some detergents. Histograms of orange fluorescence (PE) of P. aeruginosa ATCC 33351 cells incubated with biotinylated antibody antiECA898 and PE-conjugated streptavidin after preincubation in hybridization buffer containing 0.9 M NaCi and 20 mM Tris/pH 7.2 (A), and additionally 35% formam ide (B), 0.05% Tween 20 (C), 0.05% Triton X-lOO (E), or 0.05% SDS (F). Cells incubated with PE-conjugated streptavidin alone (no anti-ECA898) exhibit some nonspecific fluorescence after pretreatment with 0.05% SDS (D). For comparison, cells that are specifically stained by antibody anti-PAE2592 are also shown (G).

G. Wallner, I. Steinmetz, D. Bitter-Suermann, and R. Amann

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Order of fluorescent in situ hybridization and immunofluorescence staining steps Since the incubation conditions for FISH (46°C, detergent, formamide) were more stringent than those for IF we performed FISH prior to IF in the beginning. After encountering problems with nonspecific immunostaining using this order (due to SDS; see above) we also successfully tried the reverse order. Using Tween 20 as detergent both FISH and IF worked equally well, independent of the order of staining steps (Fig. 2). The comparison of doublestained E. coli cells (Fig. 2C and D) to cells that were only hybridized with probe EUB338 (Fig. 2A) or only indirectly immunostained with antibody anti-EC898 (Fig.2B) showed that there was no or only a slight decrease of the FISH or IF signals caused by either second staining step.

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Identification of Bacteria by Flow Cytometry

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Fig. 3. Discrimination of three bacterial species by a combination of FISH and IF. E. coli (Eco), P. aeruginosa ATCC 33351 (Pae), and C. testosteroni (Cte) were mixed and hybridized with fluorescein-labeled probe GAM42a. Then they were incubated with biotinylated antibody anti-ECA898 followed by PE-conjugated streptavidin, and counterstained with DAPI (see table 1). In panel A forward scatter (FSC) as a measure for cell size is plotted against fluorescence in the DAPI channel. No discrimination is possible. Panel B represents a plot of antibody vs. probe fluorescence, the three populations are clearly separated. Panels C and E are plots of FSC vs. probe fluorescence and panels D-F FSC vs. antibody fluorescence. In panels E and F fluorescence crosstalk of fluorescein and PE was corrected by computational compensation as compared to panels C and D.

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G. Wallner, I. Steinmetz, D. Bitter-Suermann, and R. Amann

probably the result of probe dissociation due to incubation at elevated temperature in relatively low salt buffer in their protocol (37°C in 1/10 PBS). We have also observed loss of bound probe when samples were left at room temperature for several hours. Therefore, incubation time at room temperature should be kept as short as possible, and samples should be stored on ice after staining is completed. When performing immunostaining first, A~mus et aI. (1996) found it necessary to treat the cells with methanoll acetone (1: 1) before FISH in order to make them permeable for the oligonucleotide probes. This effect occurred with several combinations of bacterial species and surface antigens (B. A~mus and M. Schloter, personal communication) and might be due to a blocking layer of antibody/ streptavidin bound to antigens expressed with high density on the cell surface that hinders probe penetration. We have not observed this effect with antibody anti-ECA898, but antibody anti-PAE2592 also caused reduced binding of probe in subsequent FISH (less than half of the normal probe signal). Therefore, for certain antibodies an additional permeabilization step between IF and FISH or the reverse order of staining steps may be advisable.

Combination of fluorescent in situ hybridization and immunofluorescence in mixed populations After optimization of the staining steps in experiments with single bacterial strains, artificial cell mixtures were analyzed by triple staining with a fluorescein-labeled, rRNA-targeted probe, a biotinylated antibody detected by PE-conjugated streptavidin, and the DNA-specific dye DAPI. For example, a mixture of E. coli, P. aeruginosa, and C. testosteroni cells (Fig. 3; Table 1) was hybridized with a probe specific for the gamma-subclass of the Proteobacteria and subsequently stained with biotinylated antibody anti-ECA898 and PE-conjugated streptavidin. Finally, the sample was counterstained with DAPI in order to distinguish cells from other particles and instrument noise. In Fig. 3B-F only those cells are plotted which are DAPI-positive and lie within the gate shown in Fig.3A. The different species are best discernible when fluorescein fluorescence (probe signal) is plotted versus PE fluorescence (antibody signal). In order to facilitate the discrimination of those false positive cells which appear

brighter than unstained cells due to fluorescence crosstalk, computational spectral compensation was applied to subtract overlapping fluorescence from the unwanted fluorochrome (see Materials and Methods; Fig. 3E and F). According to the classification by their FISH and IF signals the three cell types are presented in different grey levels. Even though immunostaining of part of the E. coli cells is not complete (up to 5% of the cells, even without FISH: compare Fig. 2) almost all cells can be identified unambiguously by probe and antibody fluorescence.

Conclusions In this work we have demonstrated that rRNA-targeted probes and antibodies can be combined for the highly specific and automated identification of micro-organisms by flow cytometry. Although for many studies FISH or IF alone may be sufficient, dual color analysis using a combination of nucleic acid probes and antibodies with different fluorochromes as labels offers several advantages. If both types of probes have mutually exclusive specificities two different taxa can be identified simultaneously in one sample. Overlapping specificities of two probes may even allow the simultaneous identification of three different taxa (d. Amann, 1995), reducing the number of samples that have to be measured. Additionally, in microscopic studies further information concerning spatial distribution can be obtained by dual color analysis, e. g., close association of different taxa or colocalization in certain microenvironments. The main benefit of the concomitant use of two specific probes for flow cytometry lies in the enhanced discrimination of positive cells from negative cells and background particulates. For applications in which background of autofluorescent particles or nonspecific probe binding pose major problems, probes with the same or overlapping specificities will increase the accuracy and reliability of specific counts if only double-stained cells are considered positive. On the other hand, counting of both mono- and double-labeled cells with such a set of nucleic acid and immuno-probes can help to reduce the number of those false negative identifications that are due to low cellular ribosome content or cell permeability for rRNA-targeted probes and weak or unstable expression of antigens.

Table 1. List of bacterial species (E. coli (Eco), P. aeruginosa (Pae), and C. testosteroni (Cte)) and the specificities of the applied probe, antibody and DNA stain DAPI for the experiment shown in Fig. 3 Specific Reagent

Fluorescent Dye

Specificity

Bacterial Species Eco

Pae +

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fluorescein

gamma subclass of Proteobacteria

+

antibody anti-ECA898

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enterobacteria

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DAPI

DNA (all cells)

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+

Cte

+

Identification of Bacteria by Flow Cytometry Alternatively, one of the two probes can be used as a 'functional probe'. Concomitant identification by the other type of probe allows correlated analyses of cell identity and function. For example, rRNA-targeted probes may be applied to quantitate the cellular ribosome content and, accordingly, to assess the general metabolic activity or potential of individual cells. Quantitation of antigens by fluorescent immuno-probes could provide information on cell functions like specific metabolic activities (antibodies against the respective enzymes) or the presence of pathogenicity/virulence factors (fimbriae, capsules, enzymes). Multiparameter analysis being one of the main advantages of flow cytometry, we have measured light scatter and DAPI fluorescence simultaneously with fluorescein and PE fluorescence (three color fluorescence) in this study. These additional flow cytometric parameters both enable the quantitation of cellular size and DNA content, respectively, and enhance the discrimination of cells from noncellular particles and instrument background. Especially in environmental samples containing high levels of particulate background, counterstaining with a DNAspecific dye is inevitable for reasonable detection efficiencies and for determining the total number of cells which is required for relative specific counts by FISH and IF. Acknowledgements. This study was supported in part by grants from the Commission of the European Communities (HRAMI I and II, contracts BIOT-CT91-0294 and BIO II-CT943098 to R.A. and D.B.-S.) and the Deutsche Forschungsgemeinschaft (Am 73/2-3). We thank B. Af5mus for critical discussion.

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Rudolf Amann, Lehrstuhl fiir Mikrobiologie, Technische Universitiit Miinchen, D-80290 Miinchen, Germany, Tel.: +49-89-28922373, Fax: +49-89-2892-2360, E-mail: [email protected]