Original Article
Rapid and Quantitative Detection of Legionella pneumophila Applying Immunomagnetic Separation and Flow Cytometry Hans Peter Fu¨chslin,1 Stefan Ko¨tzsch,1 Hans-Anton Keserue,1,2 Thomas Egli1,2*
1
Eawag (Swiss Federal Institute for Aquatic Science and Technology), ¨ Uberlandstrasse 133, P.O. Box 611, ¨ Dubendorf CH-8600, Switzerland
2
Institute of Biogeochemistry and Pollutant Dynamics, ETH Z€urich, Z€urich CH-8092, Switzerland
Received 9 April 2009; Revision Received 14 December 2009; Accepted 15 December 2009 Additional Supporting Information may be found in the online version of this article. Grant sponsor: Federal Office for Civil Protection; Grant number: 350001621. *Correspondence to: Thomas Egli, Eawag (Swiss Federal Institute for Aquatic Science and Technology), ¨ Uberlandstrasse 133, P.O. Box 611, ¨ Dubendorf CH-8600, Switzerland Email:
[email protected] Published online 22 January 2010 in Wiley InterScience (www.interscience. wiley.com) DOI: 10.1002/cyto.a.20858 © 2010 International Society for Advancement of Cytometry
Cytometry Part A 77A: 264 274, 2010
Abstract Legionella is a pathogenic bacterium that establishes and proliferates well in water storage and distribution systems. Worldwide it is responsible for numerous outbreaks of legionellosis, which can be fatal. Despite recent advances in molecular and immunological methods, the official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only. Therefore, we developed a cultivation-independent, quantitative, and fast detection method for Legionella pneumophila in water samples. It consists of four steps, starting with (1) a concentrating step, in which cells present in one litre of water are concentrated into 5 ml by filtration (pore size 0.45 lm), (2) then cells are resuspended with sterile filtered buffer and double-stained with FITC- and Alexa-conjugated Legionellaspecific antibodies, (3) subsequently, the cells are immunomagnetically caught, and (4) finally, fluorescently labeled Legionella cells were flow cytometrically detected and quantified. The efficiency of each step was tested separately. The whole method allows detection of L. pneumophila in 180 min with a detection limit of around 500 cells/l and a recovery of Legionella cells of 52.1 % out of spiked tap water. Fluorescence microscopy and flow cytometric cell-counting correlated well. ' 2010 International Society for Advancement of Cytometry
Key terms drinking water; flow cytometry; immunomagnetic separation; immunodetection; Legionella spp.; microbeads
LEGIONELLA pneumophila is responsible for numerous outbreaks and isolated cases of legionellosis worldwide. Legionnaires’ disease is caused by inhalation of contaminated water aerosols and is lethal in 5–40% of the cases (1). Legionella spp. are found especially in aquatic biofilms, not only in nature but also in warm water plumbing systems, air conditioners, or cooling towers. Because of the negative impact on the human health, a tolerance limit for L. pneumophila in tap water was set in different countries, including Switzerland (2), to 1000 colony-forming units (CFU)/l. The official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is based on cultivation. Specific shortcomings of this detection method are the long assay time of 10 days, the detection of colony-forming cells only and that it is highly labor-intensive. In practice, this delay is unacceptably long, and a specific and sensitive fast screening method is of much interest. A fast screening method would be of particular interest for quickly testing water installation systems in public buildings such as hospitals, nursing homes, or hotels before closing them to prevent an outbreak of legionellosis. Several attempts were made to develop alternative fast detection methods. The most frequently used approach for fast detection is based on the polymerase chain reaction (PCR). However, the presence of PCR-inhibitory compounds and the inability to differentiate between living and dead cells are major
ORIGINAL ARTICLE drawbacks. Furthermore, PCR tests have limited capability for accurate bacterial quantification because the cell number assigned to a certain amount of target genes varies by one order of magnitude depending on growth phase and species (3,4). Several studies were made using immunofluorescence assays (IFA) or fluorescent in-situ hybridization (FISH) for labeling combined with fluorescence microscopy for enumeration (5–7). However, quantification by fluorescence microscopy is, despite recent advances in instrument automation and digital image analysis, still a time-consuming and labor-intensive method. Flow cytometry (FCM) has potential as an alternative method for the quantification of fluorescently labeled bacterial cells in drinking water; it is fast, accurate, and quantitative (8,9). Already in the 80’s, the detection of immunolabeled Legionella in water samples of cooling towers using flow cytometry was reported, but the early stage of the technique allowed only the detection of elevated concentrations of Legionella in the range from 104 to 106 cells/ml (10,11). Recently, a method for the immunolabeled Legionella in tap water was described using solid-phase scanning cytometry with a low detection limit of 10–100 cells/l (12). Solid-phase scanning cytometry is a technology similar to flow cytometry where cells fixed on a membrane are enumerated by automatic laser scanning. In the last few years, immunomagnetic separation (IMS), using a combination of various magnetic beads or paramagnetic particles coated with antibodies against L. pneumophila surface antigens has been introduced as a purification step before analysis to detect also cells that are hidden by debris or other cells (13). Up to now, relatively large magnetic beads were used for this purpose with diameters between 1 and 5 lm (13–16) compared to the cell size of Legionella of 0.3–0.9 lm in width and 2–20 lm in length (17). However, large beads are known to have difficulties in finding their targets, and cells are known to detach easily from the large magnetic beads due to shear forces. Large beads also interfere with immunodetection and DNA extraction and, therefore, make it necessary to dissociate bacterial cells from beads before postIMS analyses. Both lead to further cell losses. Magnetic beads of smaller size (0.05 lm), such as MicroBeads, can reduce such problems; in addition, their ratio of surface per volume is higher. Recent reports show that cells labeled with MicroBeads can be easily isolated from complex matrices such as milk, apple juice, and manure with a high recovery of 95% and that they can be directly enumerated by flow cytometry (18,19). For several years, magnetic beads of 0.05 lm have been used in the field of medical analysis (20), especially for isolation of specific cells in blood samples, whereas only the two studies cited earlier reported the isolation of microorganisms with MicroBeads. Therefore, we developed a quantitative and fast screening method for L. pneumophila SG 1 in water samples combining the magnetic separation technique with MicroBeads and flow cytometry. The proposed method includes a concentrating step starting by filtration and resuspension followed by immunolabeling and a further concentrating and purification step Cytometry Part A 77A: 264 274, 2010
with immuno-magnetobeads. Subsequently, the cells were flow cytometrically detected. The different procedure steps were first tested under laboratory conditions. In a second step, the whole method was applied to spiked tap water and the detection limit was determined.
MATERIALS AND METHODS Strains and Cultivation L. pneumophila SG1 (ATCC 33152), obtained from Oxoid (Hampshire, UK) was cultivated on ready-poured plates with selective medium BMPA (Oxoid) at 378C for at least 10 days, and one single colony of L. pneumophila SG1 cells from agar plate was subsequently resuspended in 1 ml of 0.22 lm-filtered (filter type GVWP, Millipore, Billerica, USA) phosphatebuffered saline (PBS: 9.0 g/l NaCl, 1.62 g/l Na2HPO42H2O, 0.36 g/l KH2PO4, pH 7.2). All used PBS-buffers were filtered in the described way. The concentration of L. pneumophila was determined flow cytometrically and diluted with PBS to set the defined cell concentration. Alternatively, L. pneumophila SG1 was cultivated in batch culture at 378C for 3 days in modified ATCC medium 1099, i.e., CYE (Charcoal Yeast Extract) buffered medium, in which the charcoal concentration was reduced from 2 to 0.2 g/l. The cell concentration was quantified flow cytometrically, and the cell suspension was diluted with PBS to set the defined cell concentration. Spiked tap water was prepared by adding a defined volume of L. pneumophila suspension with a known number of cells to 1 l of L. pneumophila-free tap water collected from an in-house distribution system. This tap water was tested to be free of L. pneumophila by the Swiss Reference laboratory for Legionella in Bellinzona. Escherichia coli K12 MG 1655 (ATCC 700926) was cultivated in glucose-limited batch medium at 378C for 3 days. The cells were harvested in stationary phase 4 days after inoculation. The mineral medium contained, per litre: 275 mg NH4Cl, 75 mg MgSO47H2O, 5 mg CaCl2H2O, 35 mg KCl, 1.5 mg FeCl2, 60 lg H3BO3, 100 lg MnCl24H2O, 120 lg CoCl26H2O, 70 lg ZnCl2, 25 lg NiCl26H2O, 15 lg CuCl22H2O, 25 lg Na2MoO42H2O, 5.2 mg EDTANa4(H2O)4. After heat sterilization and cooling down to room temperature, 1 l of mineral medium was supplemented with glucose (98%, Fluka) as the only source of carbon and energy (1.0 g/l), 100 ml of phosphate buffer (Na2HPO42H2O/KH2PO4, 0.56 M with respect to phosphate, pH 7.5), and 0.05 ml of vitamin stock solution with the composition according to Egli et al. (21) by 0.22 lm sterile filtration using sterile disposable filters (Millipore). Filtration and Resuspension Three different types of filters made of polyvinylidene fluoride (PVDF, catalog number HVLP 02500, Millipore), teflon surface-modified membrane filters (catalogue number FHLC 02500, Millipore), and PC track etch membrane filters (type 230, Sartorius, Go¨ttingen, Germany) were evaluated to find the filter with the highest recovery. All filters had a pore size of 0.45 lm and a diameter of 25 mm. Filters were placed in a fil265
ORIGINAL ARTICLE ter-holder (Sartorius) and loaded with a defined number of PBS-suspended L. pneumophila SG1 cells. Subsequently, to wash-off cells accumulated on the membranes, filters were inversed and flushed with 10 ml PBS. Filtration and resuspension of cells from tap water. Tap water was prefiltered through a 30-lm nylon-net filter (Type NY30, Millipore) to separate bacteria from bigger particles. Then, 1 l of water was filtered through a PC track etch membrane filter (type 230, Sartorius). Subsequently, cells retained on the filter were washed away by flushing with 5 ml of PBS with 4.2 g/l NaCl and 0.05% Tween 80 from the other side. Filters were removed from the filter holder and placed with the washoff liquid in a 15 ml falcon tube, vortexed vigorously for 5 min, and incubated for 30 min under permanent agitation at 378C. Cell Staining Single staining with SYBR Green I. Determination of the total cell count in suspensions of pure cultures of Escherichia coli K12 were done flow cytometrically after staining with SYBR Green1 I nucleic acid gel stain (Molecular Probes, Basel, Switzerland). SYBR Green1 I (104-fold concentrated in dimethyl sulfoxide (DMSO)) was diluted 100-times in 0.22 lmfiltered DMSO. These stock solutions were stored at –208C. To a sample of 1 ml, 10 ll of SYBR Green1 I stock solution was added and incubated in the dark for at least 15 min before analysis. Where necessary, samples were diluted just before measurement with PBS, so that the concentration measured with the flow cytometer was always less than 2 3 105 cells/ml.
Single-staining with specific antibody. L. pneumophila SG1 cells were stained with cell-specific FITC-conjugated polyclonal IgG rabbit antibodies with a total concentration of 4 mg/ ml (Cat. No. ab20818, Abcam, Cambridge, UK). Cells were incubated for 60 min at room temperature with 0.3 ll of antibody and 10 ll of BSA (1 %) (Sigma, Steinheim, Gemany) per ml of sample. Double-staining with specific antibody. Cells were doublestained simultaneously with 10 ll of BSA (1 %) per ml of sample, 12.5 ll of 103 diluted polyclonal FITC-conjugated rabbit antibodies specific to L. pneumophila (Abcam, Cat. No. ab20818) and 10 ll of polyclonal rabbit antibodies specific to L. pneumophila (GeneTex, San Antonio, TX; Cat.No. GTX40943) conjugated with Alexa350 (Molecular Probes, Eugene, OR; Cat. No. A10170) per ml of sample. Conjugation was achieved following the instruction of the AlexaFluor1350 Protein Labelling Kit (22). The samples were vortexed vigorously and incubated for 60 min under permanent agitation at room temperature and protected from light. Immunomagnetic Cell Separation The MiniMACS magnetic cell separator (MS – Column for 107 positive cells/ml and MACS colloidal (iron oxide and polysaccharide) super-paramagnetic MicroBeads) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). 266
The used MicroBeads were 50 nm in diameter and were coated with monoclonal mouse anti-FITC antibodies (anti-FITC). The IMS procedure consisted of three steps: cell-labeling with L. pneumophila specific FITC-ab, magnetic-labeling with antiFITC MicroBeads, and magnetic separation. First, the standard protocol from Miltenyi was applied, which was later adapted and modified for the detection of L. pneumophila. Standard protocol. Labeling and separation was performed as follows: 0.3 ll of FITC-ab and 10 ll of BSA (1%) were added to 1 ml of cell suspension and incubated at room temperature for 30 min. After incubation, the sample was centrifuged at 1,500g for 6 min, and the supernatant (0.92 ml) was discarded. Aliquots of 20 ll of anti-FITC-antibody-conjugated paramagnetic MicroBeads (481-01, Miltenyi) were added and incubated for 15 min at 48C protected from light. The separation column was placed in the magnet and operated according to the manufacturer’s instructions: the column was preconditioned with 2 ml of PBS, and the eluate was discarded. Then, the sample (100 ll) was run through the column. Subsequently, the column was washed with 2 ml of PBS and the eluate was discarded. The column was then removed from the magnet, and retained cells were washed off with 1 ml PBS pressed through the column with a piston. This eluate was collected as Legionella-positive fraction and was later flow cytometrically analyzed. Modified protocol. In the modified procedure, centrifugation steps were avoided because they led to nonreproducible cell loss and the sample volume was changed from 100 ll to 10 and 5 ml, respectively. After extensive testing, the ratios of FITC/Alexa-coupled antibodies and MicroBeads were optimized and adapted to the higher sample volume. The resulting protocol for labeling, capturing, and separation is as follows: for single-staining 0.5 ll FITC-ab, and 50 ll of BSA (1%) were added to 10 ml sample. For double-staining, 0.15 ll FITC-ab, 2 ll Alexa-ab, and 50 ll of BSA (1%) were added to 5 ml sample. The solution was incubated at room temperature for 60 min. Then, 100 ll of antiFITC-ab-conjugated paramagnetic MicroBeads were added and incubated for 30 min at 48C protected from light. The separation column was preconditioned with 2 ml of PBS, and the eluate was discarded. Subsequently, the extraction column was placed in the magnet and operated according to the manufacturers instructions. The sample (10 or 5 ml) was run through the column. Subsequently, the column was washed with 2 ml of PBS and the eluate was discarded. The column was then removed from the magnet, and retained cells were washed-off with 1 ml PBS pressed through the column with a piston. This eluate was collected as Legionella-positive fraction and was later flow cytometrically analyzed. Cell-counting Using Fluorescence Microscopy Spiked and labeled samples were filtered onto 0.20 lm GTPB microscopy black membrane filters (Millipore). The black membrane filter was then placed on a microscopy glass Rapid and Quantitative Detection of L. pneumophila
ORIGINAL ARTICLE Table 1. FCM cell-counting (n 5 6) was compared with standard methods such as fluorescence microscopic counting (n 5 3 filters, on each filter 10 fields of approximately 2002300 cells/field) and plate counting (n 5 9) AGE OF THE CULTURE
5 days 5 days 90 days
FLUORESCENCE MICROSCOPY (COUNTS/ML)
PLATE COUNTING (CFU/ML)
FLOW CYTOMETRY (COUNTS/ML)
RATIO (%)
799,174 75,364 – –
– 33,565 6,055 n.d.
774,200 44,040 112,648 1,696 164,380 2,104
103.23 29.80 0.00
n.d., not detected. The age of used L. pneumophila SG1 plate cultures (time after spreading) at the time of analysis was 5 and 90 days, respectively. The FCM count is defined as 100%.
slide. When air dried (after approximately 3 h), 5 ll of antifade mounting medium (Waterborne, New Orleans, LA) was added to the stained cells on the membrane, and it was covered with a cover slide suitable for fluorescent microscopy. The samples were examined within 5 h using an Olympus IX51 microscope with a 603 UPlanFI objective lense, 103 eyepieces equipped for epifluorescence microscopy and using immersion oil from Olympus appropriate for fluorescence microscopy (Olympus, Tokyo, Japan). For enumeration, the software Olympus DP-Soft, Version 3.2 was used. The bacterial cell concentration was calculated as follows: number of cells per milliliter 5 (average cell count/microscope field) 3 ((area of membrane filter)/(area of microscopic field)) 3 (dilution factor of sample)/sample volume filtered. For one measurement, more than 10 microscopic fields were counted with a minimum of 1,000 cells counted per assay (Fig. S1, Supp. Info.). Flow Cytometric Analysis Flow cytometric (FCM) was performed using a Cyflow1 space flow cytometer (Partec, Mu¨nster, Germany) equipped with two lasers, a 200 mW diode solid phase laser emitting at a fixed wavelength of 488 nm and an ultraviolet laser (9 mW, 374 nm). The cell number was enumerated in an aliquot of 200 ll of sample. Data were analyzed using Flowmax software (Partec).
Analysis of SYBR Green I-stained cells. Determination of the total bacterial cell concentration was based on the combination of green fluorescence (520 30 nm) and red fluorescence ([630) (23). The specific instrumental gain settings were as follows: green fluorescence (420), red fluorescence (755), and speed 2 (implying a counting rate of less than 500 events/s). All samples were triggered on green fluorescence. Analysis of single L. pneumophila-specific ab-stained cells. The identification of L. pneumophila was based on the combination of green fluorescence and Side Scatter (SSC) (Fig. S2, Supp. Info.). The specific instrumental gain settings were as follows: green fluorescence (420), side scatter (700), and speed 2. All samples were triggered on green fluorescence. Analysis of double L. pneumophila-specific ab-stained cells. The identification of L. pneumophila SG1 was based on the multiple gating of SSC 375 nm, SSC 488 nm, FSC 488nm, Cytometry Part A 77A: 264 274, 2010
fluorescence at 445, 520, and 630 nm. The specific instrumental gain settings were as follows: UV fluorescence 375–455 nm (474), side scatter 375–375 nm (435), green fluorescence 488– 520 nm (535), red fluorescence 488–630 nm (786.5), side scatter 488–488 nm (303), foreward scatter 488–488 nm (173.5); the speed was 2 (implying a counting rate of less than 500 events/s). All samples were triggered on UV fluorescence 375– 455 nm. Enumeration of Legionella sp. by Plating Legionella sp. cells were enumerated according to the ISO-11731 standard method directly, omitting acid treatment, heat treatment, and centrifugation. Statistical Analysis All measurements were repeated at least three times; standard deviations were calculated and are shown. All statistical analyses were performed using Microsoft1Office Excel, 2003. Standard methods to calculate descriptive statistics were used. The association between microscopic and flow cytometric counting was tested using a t-test (P values are two-sided). For testing the linear relationship, the two-sided P-value for a given correlation value r and sample size was calculated.
RESULTS Comparison between FCM and Fluorescence Microscopy Cell-counting A suspension of L. pneumophila SG 1 cells in PBS was stained with specific FITC-coupled antibodies and enumerated by both fluorescence microscopy and flow cytometry. A fluorescence microscopy picture, typical flow cytometric histograms, and dot plots are shown in Figures S1 and S2 (Supp. Info.). The fluorescence microscopy and the flow cytometric cell-counting correlated well (103.23 %, P 5 0.44) defining the FCM cell count as 100% (Table 1). However, the relative standard deviation (% RSD) of flow cytometric enumeration (5.69%) was significantly lower than that for enumeration by fluorescence microscopy (9.43%). FCM Cell-counting Compared to Plate-counting Flow cytometric enumeration of FITC-coupled antibodies stained L. pneumophila SG 1 was compared with the standard plate-counting of a 5-day and a 90-day-old culture. Compared to the flow cytometric enumeration, the plating efficiency was in the case of the 5-day-old culture with 29.80% compared to the FCM cell count already quite low and in the 267
ORIGINAL ARTICLE Determination of the Flow Cytometric Detection Limit The FCM detection limit was determined by measuring a dilution series of FITC-conjugated ab-stained L. pneumophila SG 1 cells grown on plates and resuspended in PBS. A good linear regression (R2 5 0.99) of the total calibration series was observed (Fig. 1a), and the linear regression of 0.98 extended down into the range between 0 and 250 cells/ml (Fig. 1b). In general, the detection limit was defined as the lowest cell concentration that can be distinguished from the negative control within a defined confidence limit. We defined the lowest positive signal as detection limit, at which the threefold standard deviation bars do not interfere with the threefold standard deviation bar of the negative control. On the basis of this criterion the detection limit of FCM ‘‘Cyflow1 space’’ for immunostained L. pneumophila SG 1 cells was determined as 107 counts/ml (Fig. 1b). The average of all %RSD was 10.8%.
Figure 1. (a) Calibration series of FITC-conjugated ab-stained L. pneumophila SG1 cells detected in 1 ml of PBS. The spiked cells concentration in spiked samples is shown versus the concentration of FCM-detected L. pneumophila SG1 cells. All samples were measured in triplicate. The black line represents the linear regression function (y 5 1.0832x — 112.23, R2 5 0.9998, P \ 0.000001) and error bars are indicating standard deviations. (b) Zoom into the 0—250 cells/ml concentration range of (a). The black line indicates the linear regression function for the presented data points (y 5 0.9344x 1 10.25, R2 5 0.9835, P 5 0.0009). Y-Error bars indicate the threefold standard deviation. The detection limit of 107 L. pneumophila SG1 cells/ml is defined by non-overlapping threefold standard deviation of a sample with the negative control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com]
case of the 90-day-old culture, no cultivable cells were detectable at all (Table 1). In contrast, the flow cytometric signals obtained for cells from the 5-day-old and the 90-day-old culture had approximately the same fluorescence intensity. Flow cytometric enumeration was statistically more reliable, i.e., the % RSD was with 1.51% significantly lower than that of the plating method (18.04%). The results indicate that the plating method is much more prone to physiological changes of the cells than the flow cytometric enumeration method. 268
Concentrating by Filtration and Resuspension The first concentrating step of the proposed method consists of a collection of bacterial cells present in the water sample on a membrane filter and their subsequent resuspension in a smaller volume. Three different filters (PVDF, Teflon surface-modified, and PC track etch membranes) were evaluated to find the filter with the highest recovery. All filters had a pore size of 0.45 lm and all were loaded with 30,000 L. pneumophila SG 1 cells suspended in PBS. A total of 30,000 L. pneumophila SG1 cells were spiked into 100 ml PBS, collected on different membrane filters and subsequently resuspended with 10 ml of PBS. The recovery was defined by flow cytometric counting of antibody-stained L. pneumophila SG 1 cells of the original and the resuspended sample. The results for the recovery obtained with the three different filters after this procedure are shown in Table 2. The recovery with PVDF and Teflone membrane filters was in the range of 50%, recoveries obtained with the PC track etch membrane filters were consistently around 90%. Therefore, PC track etch membrane filters were used in further experiments with three different cell concentrations, where an average recovery of 89.18% 1.84% was obtained (Table 2). Table 2. Recovery of 30,000 L. pneumophila SG1 cells spiked into 100 ml PBS after filtration onto three different filter membranes (n 5 3) and resuspension with 10 ml PBS TYPE OF FILTER
PVDF filter Teflon filter surface modified Polycarbonate track etch membrane filter
SPIKED CELL NUMBER (CELLS)
RECOVERY (%)
30,000 333 30,000 333
43.85 2.82 53.90 7.50
30,000 333
87.09 1.22
247,383 3481 22,617 1478 3,400 234
87.19 2.14 89.54 1.11 90.82 0.56
In addition, the filter with the best recovery was tested (n 5 3) with three different cell numbers.
Rapid and Quantitative Detection of L. pneumophila
ORIGINAL ARTICLE Table 3. Immunomagnetic separation of single FITC-stained L. pneumophila SG1 from 10 ml of PBS with standard method defined by the manufacturer and modified procedure adapted for the separation out of water samples with higher volumes NUMBER OF L. PNEUMOPHILA CELLS IN 10 ML PBS (COUNTS)
RECOVERED BY FCM L. PNEUMOPHILA SG1 (COUNTS)
5,840 230 5,342 56 42,527 585 38,158 1,114 402,930 7,288 376,338 4,781 Modified 0 257 104 method 4,544 623 4,246 177 40,330 902 39,152 653 370,684 18,712 346,626 10,191 Original method
RECOVERY (%)
91.47 3.83 89.73 1.72 93.40 1.25 – 87.78 3.90 96.44 1.62 93.44 2.94
Measurements were made in triplicate. Recovery was defined as the ratio between flow cytometric cell counts after immunomagnetic separation subtracted the cell counts in the negative control and the flow cytometric cell counts before immunomagnetic separation.
Immunomagnetic Cell Separation with MACS1, Original and Modified Procedure The second step of the proposed method consists of purification and further concentrating of the cells by immunomagnetic separation. The recovery was defined as the ratio between the cell number of immunomagnetically recovered cells and the original number of cells in the sample before immunomagnetic cell separation determined by flow cytometric enumeration of specifically immunostained cells. First, the original procedure as described by the manufacturer was applied using three different cell concentrations in triplicate. The recovery for all cell concentrations tested was on average 91.53% 2.26% (Table 3). In the modified immunomagnetic separation method, centrifugation steps were omitted to avoid cell loss and the sample volume was changed to 10 ml to accommodate the filtration/resuspension procedure. Antibody and MicroBead concentration were optimized for the new sample volume. The results for the immunomagnetic separation experiments with three different cell concentrations ranging over two orders of magnitude and a negative control, all conducted in triplicate, are shown in Table 3. The recovery over the whole concentration range was on average 94.68% 2.82%. The number of false-positive counts in the negative control was on average 257 104. The FCM signal before and after cell
separation was very similar, indicating that the attached MicroBeads did not significantly disturb FCM detection (Fig. S3, Supp. Info.). The free FITC-conjugated ab, which were not bound to L. pneumophila cells, were probably bound to free anti-FITC MicroBeads and they yielded a signal in the low range of green fluorescence (520 nm) and a strong signal in side scatter (SSC) (Figs. S3b and S3c, Supp. Info.). This suggests that unbound FITC-conjugated ab agglutinate together with unbound anti-FITC MicroBeads and form clusters. Immunomagnetic Cell Separation from a Defined Mixed Population L. pneumophila SG1 was immunomagnetically separated from a defined mixed population of E. coli and L. pneumophila SG1 (Table 4). For this purpose, three aliquots containing different amounts of cells of L. pneumophila SG1 cells were mixed with 3.66 3 106 cells of E. coli K12 in 10 ml of PBS. The recovery of FITC-conjugated ab stained L. pneumophila SG1 was on average 92.23% 1.01%. The number of false-positive counts in the negative control (278 108) was similar to the falsepositive counts for the pure culture (257 104). In control experiments, virtually all E. coli K12 cells were found in the negative fraction (98.73% 0.71%). Double Staining of Pure Culture The first attempt failed to detect singly-labeled L. pneumophila, immunomagnetically separated from spiked tap water. It was not possible to discriminate the singly labeled Legionella from the background (data not shown). Therefore, it was decided to apply a double-staining to L. pneumophila with two different antibodies conjugated with different stains (Alexa 350 and FITC). Figure S4 (Supp. Info.) shows the double-stained L. pneumophila cell fluorescence in green and blue color under the microscope. High recovery of more than 95% was achieved by single-labeling and detection at the appropriate wavelength. Different staining procedures (simultaneous and sequential) were tested for double-staining (Table 5). The highest recovery was obtained by simultaneous incubation with both antibodies. In several experiments, the best concentrations of the two antibodies were determined, that gave highest recovery. Double-staining of Spiked Tap Water The optimized double-staining method was tested with spiked tap water: Cells from 1 l of spiked tap water were con-
Table 4. Immunomagnetic separation of FITC-stained L. pneumophila SG1 from a defined mixed culture of E. coli K12 and L. pneumophila SG1 in 10 ml of PBS applying the modified immunomagnetic separation method with single-staining E. COLI (CELLS IN 10 ML)
L. PNEUMOPHILA SG1 (CELLS IN 10 ML)
RECOVERED L. PNEUMOPHILA SG1
RECOVERY
3,668,000 44,594 3,668,000 44,594 3,668,000 44,594 3,668,000 44,594
0 3,426 68 34,383 383 344,563 7,565
278 108 3,130 158 31,628 2,424 321,615 10,268
– 83.25 4.61 91.18 7.05 93.26 2.98
Recovery was defined as the ratio between specific flow cytometric cell counts for L. pneumophila after immunomagnetic separation subtracted the cell counts in the negative control and before immunomagnetic separation.
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Table 5. Comparison of different staining procedures STAINING PROCEDURE
CELL COUNTS (375 NM)
CELL COUNTS (520 NM)
CELL COUNTS MULTIGATING (375/520 NM)
Single-staining FITC-ab Single-staining Alexa 350-ab Double-staining sequential 1. FITC-ab 2. Alexa 350-ab Double-staining sequential 1. Alexa 350-ab 2. FITC-ab Double-staining simultaneously Alexa 350-ab/FITC-ab
n.d. 102.78% 0.34% 88.94% 0.71%
100.00% 2.03% n.d. 98.50% 1.55%
n.d. n.d. 89.77% 0.86%
99.35% 2.31%
96.40% 3.87%
90.36% 6.91%
101.45% 1.61%
98.80% 4.68%
97.69% 1.44%
n.d.: not detected. A suspension of 300,000 L. pneumophila SG1 cells per ml PBS were stained differently (single-staining versus double-staining, simultaneous staining versus sequential staining) and detected at different emission wavelengths. The simultaneous double-staining gave the highest recovery by multigating.
Figure 2. Flow cytometrical histograms and dot plots of Alexa 350 and FITC double-stained L. pneumophila SG1 cells after immunomagnetic separation with MACS1 MicroBeads from 1 l of spiked tap water. A positive control of 300,000 cells spiked in 1 l of tap water (a) and a negative control (b) are shown. Multiple logical gating (R1 AND R2 AND R3 AND R4) was used for enumeration purposes, allowing distinction between the background and the double-stained cells. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com]
ORIGINAL ARTICLE centrated by membrane filtration and resuspended again in 5 ml of PBS with 4.2 g/l NaCl and 0.05% Tween 80. The suspension was incubated with the two antibodies and then cells were immunomagnetically separated. Finally, the enriched and cleaned L. peumophila cells were flow cytometrically quantified. Typical flow cytometric histograms and dot plots of a positive and negative sample are shown in Figure 2. The FCM detection limit was determined by measuring a series of spiked tap waters with different L. pneumophila concentrations. A good linear regression (R2 5 0.99) of the total calibration series was observed (Fig. 3a) and the linear regression of 0.98 extended down into the range between 3,000 and 0 cells/l (Fig. 3b). The detection limit was defined as the cell concentration at which it is still possible to statistically significantly distinguish between positive signal and negative control; this was defined as the lowest positive signal, at which the threefold standard deviation bars did not interfere with the threefold standard deviation bar of the negative control. On the basis of this criterion, the detection limit of FCM ‘‘Cyflow1 space’’ for double-immunostained L. pneumophila SG 1 cells was determined as 500 cells/l (Fig. 3b). The recovery of every procedure step was checked by microscopic and flow cytometric counting (Table 6). A good correlation was found between microscopic and flow cytometric results. The most significant loss of cells was found to occur during the filtration and resuspension step (32.6%).
DISCUSSION A method was developed that allows rapid and quantitative detection of L. pneumophila in water samples. The method comprised concentrating by filtration and resuspension, immunostaining followed by immunomagnetic separation using labeling with paramagnetic MicroBeads (size 50 nm), separation on a high-gradient column, and finally flow cytometric detection. The individual steps of the procedure were separately validated under laboratory conditions, and the results were compared with established standard methods such as cell enumeration with fluorescence microscopy and colony-forming units on selective agar plates. Furthermore, the whole method was tested with spiked tap water, and the detection limit was determined. In a first step, different membrane filters were evaluated for concentrating and highest recovery of around 89.18% 1.84% was obtained reproducibly with polycarbonate track etch membrane filters. A similar, although slightly lower recovery of 73% for bacterioplanktonic cells in marine samples with polycarbonate membrane filters was reported by Sekar et al. (24). A second important step in the procedure, very much susceptible to cell loss, was the binding of cells to immunomagnetic beads. Critical was here the size of the beads used. The efficiency of immunomagnetic binding and subsequent separation was significantly higher with MicroBeads than with the larger magnetobeads. The use of Dynabeads M280, which have a diameter of 280 nm, was reported to give a recovery of 59.9% in distilled water (25), which is considerably lower compared to the average recovery rate of 92.2% of L. pneumophila in our experiments with MicroBeads. In general, Cytometry Part A 77A: 264 274, 2010
Figure 3. (a) Calibration series of doublestained FITC-ab and Alexa350-ab-stained L. pneumophila SG1 detected in 1 ml PBS. The spiking level is shown versus FCM-detected L. pneumophila SG1 cells (measured in triplicate). The solid line is presenting the linear regression function (y 5 0.5206x 1 120.65, R2 5 0.9971, P \ 0.000001) and error bars are indicating standard deviations. (b) Zoom into the 0—3,000 cell number concentration range of (a). The solid line is indicating the linear regression function for the presented data (y 5 0.5832x — 808.31, R2 5 0.9951, P \ 0.000001). Y-Error bars indicate three-times the standard deviation. A detection limit of 500 L. pneumophila SG1 cells/l was defined applying a statistical security of three standard deviations, i.e., the value of 500 L. pneumophila SG1 cells/l is the lowest spiking concentration where its threefold standard deviation does not interfere with the threefold standard deviation of the negative control. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com]
better recoveries have been reported for MicroBeads compared to bigger beads. For example, in earlier studies where MicroBeads were applied for separation and purification for Cryptosporidia oocysts and Listeria cells, recoveries between 67 and 95% were obtained (18,19). In comparison studies where Dynabeads were used for the detection of Mycobacterium avium, Campylobacter jejuni, or Cryptococcus neoformans, recoveries were on average lower, i.e., in the range of 47–87% (14–16). It seems that MicroBeads find their target better and, in the case of Legionella, the direct analysis with flow cytometry avoids the loss of cells in a detachment step. 271
ORIGINAL ARTICLE Table 6. Recovery of different procedure steps determined by fluorescence microscopic L. pneumophila cell counting (n 5 3 filters with 10 count pictures) and flow cytometry cell counting (n 5 3)
MATRIX
PROCEDURE STEPS
10 ml spiked filtered PBS 10 ml spiked filtered water 1 l spiked filtered tap water
1 l spiked tap water
Detection Detection Filtration Resuspension Detection Filtration Resuspension IMS Detection
RECOVERY
FLUORESCENCE MICROSCOPY (CELLS)
AGREEMENT FCM/FM
150,006 4,862 125,767 880 101,163 3,522
100.0% 3.2% 83.8% 0.7% 67.4% 3.5%
154,555 22,904 124,798 20,025 98,227 6,659
97.2% 4.6% 103.3% 19.1% 103.5% 8.9%
73,563 6,970
49.0% 9.5%
65,717 5,289
112.7% 11.2%
FLOW CYTOMETRY (CELLS)
Without previous concentration, a detection limit of 107 L. pneumophila cells/ml was obtained, which is clearly below the detection limits of 104–106 cells/ml determined in flow cytometrical studies some 20 years ago (10,11). In our research group, similar detection limits of 200 cells/ml were reached for SYBR green-stained cells of natural bacterial flora in drinking water (23). Other studies reported detection limits for immunostained cells in the range between 104 and 102 cells/ml (26–28). Finally, the whole method was tested with tap water spiked with L. pneumophila SG1 grown under laboratory conditions in suspensions. When labeling of cells with a single fluorescent antibody, it was not possible to detect spiked L. pneumophila cells ‘‘fished’’ from tap water. The positive signal of the singly-stained L. pneumophila SG1 cells was covered by a huge background noise. Attempts failed to separate the positive signal from the background by staining the DNA unspecifically either with DAPI or Hoechst 33342, besides the labeling with FITC-conjugated antibody; the UV-signal of the stained DNA was too weak for our detector (data not shown). Therefore, we decided to double-stain the Legionella cells with two different antibodies, each of them coupled to a different fluorescent stain either FITC or Alexa 350 emitting at different wavelengths. A similar strategy was already reported for the detection of Cryptosporidium sp. (29). Double-staining did not extend the time for the whole procedure. The two lasers of the flow cytometer employed excite the double-stained Legionella cells at 375 and 488 nm provoking a double set of scattering and fluorescencing signals. By multiple gating of the different signals (SSC 375 nm, SSC 488 nm, FSC 488nm, fluorescence 445, 520, and 630 nm), it was possible to separate the positive signal from the background. Double-staining allows
the detection of L. pneumophila in 180 min with a detection limit of about 500 cells/l and a recovery of 52% (Table 7). Detection of L. pneumophila cells in tap water was generally possible, with the exception of water from strongly corroded pipes (data not shown). Most probably, iron oxides disturbed the magnetic separation and improved detection of L. pneumophila might be possible by eliminating iron oxides previous of immunomagnetic separation. Immunostaining of L. pneumophila in environmental samples is a challenge involving several problems. In our experience, the most important ones were as follows: first, if the applied antibodies are not specific enough they can cross-react with similar epitopes leading to false-positive results because in tap water a wide range of different organisms are present. According to the manufacturers, cross-reactions cannot be excluded for the currently used polyclonal FITC-conjugated antibodies for L. pneumophila (30,31). A second problem, also related to immunology, that may be encountered in environmental samples is that cell surface epitopes might differ for naturally grown or environmentally stressed L. pneumophila from those expressed by laboratory strains grown on agar medium, as it was shown for E. coli O157 (32). For a future improvement of the method, it will be crucial that highly specific monoclonal antibodies for the naturally grown pathogens are available. Alternatively, the recent developments in the production of artificially designed ‘‘antibodies’’ might open new opportunities (33). At present, no Legionella-specific antibody-coated MicroBeads are commercially available and, therefore, we had to apply an indirect method by using anti-FITC MicroBeads. A direct method would greatly simplify the procedure and in
Table 7. Recovery and needed time for the individual process steps
Recovery Time
FILTRATION AND RESUSPENSION STEP
STAINING AND IMMUNOMAGNETIC SEPARATION STEP
FLOW CYTOMETRIC DETECTION
COMPLETE PROCEDURE
67.4% 45 min.
81.6% 130 min.
97.7% 5 min.
52.1% 180 min.
The experimentally determined recovery of the whole procedure (52.1%) and the calculated recovery based on the single steps (67.4% 3 81.6% 3 97.7% 5 53.7) are virtually the same.
272
Rapid and Quantitative Detection of L. pneumophila
ORIGINAL ARTICLE combination with double-staining, one might be able to distinguish Legionella from other bacterial cells based on a labeling with three surface antibodies (one coating antibody of the MicroBeads and two surface-specific antibodies conjugated with different fluorescence stains). This would greatly reduce the potential for false-positives. For attaining highest specificity, the three antibodies should be monoclonal and each of the antibodies should be specific for a separate cell-surface epitope. However, highly specific monoclonal antibodies will attach to less epitopes on the bacterial surface than polyclonal antibodies with a broader spectrum of suitable antigens. This requires the use of stronger fluorescence stains such as quantum dots (34), to obtain a clear-cut separation of positive and negative cells even for cells bound to only a low number of fluorochrome-labeled antibodies. Of course, our method does not allow the detection of Legionella cells that are integrated in a biofilm or located within amebae. Nevertheless, with amebae-specific antibodies the method could be used to detect the protozon hosts itself. Furthermore, our method does not distinguish between living and dead cells; however, the smooth immunomagnetic separation enables that the detection can be coupled with the determination of the physiological state of the cells, for example, by life-dead staining (35), the BacLight bacterial viability kit, or the carboxyfluorescein diacetate assay (36). According to a recent study, a large proportion of Legionella sp. in man-made environments is in a viable-but-nonculturable state (37). It was shown that nonculturable Legionella sp. can become culturable again and still keep their pathogenity for chick embryos (38). Hence, both studies emphasise the importance of detecting also L. pneumophila-cells in the ‘‘viable-but-nonculturable state.’’ Therefore, our FCM assay that detects Legionella cells also in a viable-but-non-culturable state might give probably more realistic numbers than the standard ISO-plating procedure (Table 1). The proposed fast screening method can be applied in principle to every bacterium or parasite if specific antibodies are available that yield a strong enough signal for the detection by flow cytometry. The MicroBeads allow a fast, efficient separation even from complex matrices (18,19). This could be of interest especially for application in food and medical microbiology, where cells have to be isolated from complex matrices before detection. After immunomagnetical separation the cells can be analyzed not only by flow cytometry but also by molecular methods such as PCR. This would result in short analysis times for a double-proofed method relying on two different methodological approaches. Technological progress will lead to better fluorescence dyes, stronger lasers, more precise optics, and more sensitive detectors and we believe, therefore, that flow cytometry in combination with immunomagnetic separation will be an interesting alternative to standard plating methods in the near future.
ACKNOWLEDGMENTS The work was funded by the Federal Office for Civil Protection (Project-Nr. 350001621) and we thank SPIEZ LABORATORY for their support, in particular Nadia Schu¨rch and Cytometry Part A 77A: 264 274, 2010
Martin Schu¨tz. We also thank Dr. Valeria Gaia from the Swiss Reference laboratory for Legionella for testing tap water samples and scientific contributions.
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Rapid and Quantitative Detection of L. pneumophila
