Environmental Microbiology Reports (2010) 2(1), 158–165
doi:10.1111/j.1758-2229.2009.00127.x
Detection of environmental Vibrio parahaemolyticus using a polyclonal antibody by flow cytometry emi4_127
Anita Manti,1 Tania Falcioni,2 Raffaella Campana,3 Davide Sisti,1 Marco Rocchi,1 Vicente Medina,2 Sabrina Dominici,4 Stefano Papa1 and Wally Baffone3* 1 Department of Human, Environment and Nature Sciences, 3Department of Biomolecular Science, Division of Toxicological, Hygiene and Environmental Sciences and 4Department of Biomolecular Science, Division of Biochemical and Biomolecular Sciences, University of Urbino ‘Carlo Bo’, Urbino, Italy. 2 Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida, Lleida, Spain. Summary The aim of this study was to detect and quantify Vibrio parahaemolyticus using flow cytometry (FCM) in combination with a polyclonal antibody developed in our laboratory. Experiments were carried out using V. parahaemolyticus cells in pure and mixed bacteria culture suspensions in either artificial or natural seawater. Using FCM, V. parahaemolyticus cells labelled with the polyclonal antibody and a secondary fluorescein isothiocyanate-conjugated antibody were detected and rapidly quantified at low cell densities (103 cells ml-1) in both the pure and mixed cultures. To determine the specificity of our antibody, its crossreactivity with other ATCC bacterial strains and some environmental Vibrio spp. and Gram-positive isolates was also assessed. Significant immunoreactivity levels above background were obtained for V. harvey 64, V. parahaemolyticus 704 and V. alginolyticus 1407, although the intensities were significantly less than for V. parahaemolyticus Conero. The experiments carried out in natural seawater confirmed the antibody specificity towards V. parahaemolyticus Conero even if a lower proportion of labelled cells was observed. The application of FCM in combination with a primary polyclonal antibody appears to be a promising technique for the detection and quantification of V. parahaemolyticus cells in aquatic environments. Received 28 August, 2009; accepted 18 November, 2009. *For correspondence. E-mail
[email protected]; Tel. (+39) 722303543; Fax (+39) 722303541.
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Introduction Vibrio parahaemolyticus is a Gram-negative halophilic bacterium found in estuarine waters worldwide and it is recognized as being a leading cause of food-borne gastroenteritis (Potasman et al., 2002; de Sousa et al., 2004; Hayat Mahmud et al., 2006). An increasing number of V. parahaemolyticus outbreaks of gastroenteritis have been observed across the world since 1996 caused by diverse serotypes (Okuda et al., 1997; Bag et al., 1999; Chiou et al., 2000; Chowdhury et al., 2004; Ansaruzzaman et al., 2005); moreover, a number of cases of V. parahaemolyticus infections have also been reported more recently in Europe (Martinez-Urtaza et al., 2004). Clinical strains of V. parahaemolyticus are currently detected and identified using biomolecular techniques that test for the presence of the virulence genes tdh and trh (Matsumoto et al., 2000; Okura et al., 2003) which, respectively, encode thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) (Okuda et al., 1997; Park et al., 2004). On the other hand, the detection and identification of environmental strains is generally performed by biochemical and microbiological tests which are time consuming and require additional confirmation through the use of biomolecular methods, such as ribotyping (Bag et al., 1999; DePaola et al., 2003; Gendel et al., 2001), pulsed field gel electrophoresis (Bag et al., 1999; Marshall et al., 1999) and PCR (Okuda et al., 1997; Matsumoto et al., 2000; Hara-Kudo et al., 2003; Baffone et al., 2006). It has also been shown that the detection of V. parahaemolyticus by PCR is specific and less time consuming than conventional bacteriological methods (Brasher et al., 1998; Bej et al., 1999); however, to achieve the desired detection sensitivity of 101–102 cfu g-1, enrichment cultures must be employed. Immunological methods are highly sensitive and can accurately detect several bacterial species (Hegarty et al., 1999; Rengpipat et al., 2008). Until now, however, very few studies have reported the production and use of diagnostic antibodies for the identification of V. parahaemolyticus isolates (serovars), and some of which also report the occurrence of antibody cross-reactivity (Chen et al., 1992; Chen and Chang, 1996; Datta et al., 2008).
Antibody-labelled V. parahaemolyticus detected by FCM 159 Flow cytometry (FCM) in combination with fluorescent probe technology has been successfully applied for the rapid and specific detection and enumeration of bacteria in medical, veterinary and environmental microbiology (Porter et al., 1993; McClelland and Pinder, 1994; Kusunoki et al., 1998; Chitarra et al., 2002), but, to our knowledge, no studies have reported its use for the detection of V. parahaemolyticus in aquatic environments. Flow methods permit the direct examination of seafood or water and do not involve lengthy enrichment steps or overnight incubation, thus rendering the detection and enumeration V. parahaemolyticus cells an easier and less time-consuming process. In this study, FCM used in combination with a primary polyclonal antibody and secondary fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody was evaluated for its ability to detect and quantify V. parahaemolyticus cells. The aims of this research were as follows: first, to test the sensitivity and specificity of our antibody in pure suspensions of V. parahaemolyticus and in suspensions containing several food- or water-borne pathogens that usually live in aquatic environments; second, to detect V. parahaemolyticus in both artificial and natural seawater.
Results and discussion Antibody labelling in FCM Vibrio parahaemolyticus Conero, an environmental strain isolated from molluscs in the Adriatic Sea and first identified using the method proposed by Alsina and Blanch (1994), was used to validate a new cytometric system to detect V. parahaemolyticus using the primary polyclonal antibody and a secondary FITC-conjugated antibody reported in our previous study (Falcioni et al., 2005). The detection procedure was carried out for V. parahaemolyticus in: (i) sterile artificial seawater (ASW: NaCl 24.7 g l-1, KCl 0.67 g l-1, CaCl22H2O 1.36 g l-1, MgCl26H2O 4.66 g l-1, MgSO47H2O 6.29 g l-1, NaHCO3 0. 18 g l-1; Wolf and Oliver, 1992); (ii) seawater filtered through a 0.22 mm filter and (iii) natural seawater. In order to determine the total cell count, aliquots of each sample were labelled with the fluorochrome SYBR Green I (Molecular Probes) that has a high affinity for nucleic acids. Cytocount™ counting beads (DakoCytomation) were added to the stained samples and processed by the flow cytometer (Manti et al., 2008). Flow cytometry analysis, performed using a FACScalibur flow cytometer (Becton Dickinson), showed that the
Fig. 1. Relative proportion (ordinate axis) of labelled cells for each bacterial strain (abscise axis). Mean proportions with 95% confidence interval (CI) lines are shown. Dots represent mean proportions; bars indicate CI. Bacterial cell suspensions (106 cells ml-1) were incubated at a ratio of 1:200 with our primary polyclonal antibody for 30 min at room temperature (RT) in 100 ml of PBS. Samples were then washed twice in PBS at 3000 r.p.m. for 15 min and labelled with FITC-conjugated anti-mouse IgG (Sigma) at a ratio of 1:1000 for 30 min in the dark at RT (Falcioni et al., 2005).
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 158–165
160 A. Manti et al. environmental samples can never be unequivocally demonstrated (Hegarty et al., 1999). The possibility of cross-reactivity of the antibody with an organism other than V. parahaemolyticus must therefore also be considered. Cross-reactivity of the V. parahaemolyticus Conero polyclonal antibody against other ATCC bacterial strains (V. parahaemolyticus ATTC 43666, V. alginolyticus ATCC 33787, Escherichia coli ATCC 8739, Lysteria monocytogenes ATCC 7644, Aeromonas hydrophila ATCC 7966, Salmonella enteritidis ATCC 13076, Staphylococcus
Fig. 2. Electron micrographs of V. parahaemolyticus Conero (A and B) and E. coli ATCC 8739 (C and D) labelled with our primary polyclonal antibody and gold-conjugated secondary antibody. Arrows point to gold particles. Bars = 0.58 mm. Bacterial cells were fixed in 4% paraformaldehyde in PBS, dehydrated in alcohol and processed for cold-embedding in Lowicryl K4M resin (Wells, 1985; Caciagli et al., 2009). After polymerization, ultrathin sections (~95 nm) were cut and collected on nickel grids. Sections on grids were incubated for 1 h in blocking buffer, washed in distilled water and incubated for 3 h at RT in the polyclonal antibody specific against V. parahaemolyticus Conero cells. After further incubation in blocking buffer and washing, grids were incubated for 1 h at RT in goat anti-mouse antibodies conjugated to 10 nm gold particles (Janssen AuroProbe; Janssen Pharmaceuticals). Finally, the stained grids were observed in a transmission electron microscope (EM 910, Zeiss).
anti-V. parahaemolyticus Conero polyclonal antibody specifically binds to its target (Fig. 1). The confidence interval (CI) for each proportion was evaluated after performing arc sin√ transformations, in order to minimize heteroskedasticity. Cross-reactivity tests in FCM It is important to note that absolute specificity of antibody preparations used in the examination of
Fig. 3. Confocal microscope images of V. parahaemolyticus Conero (A) and E. coli ATCC 8739 (B) labelled with the our primary polyclonal antibody and FITC-conjugated secondary antibody; (C) phase contrast light microscope image of the same E. coli ATCC 8739 sample visualized in B. Paraformaldehyde-fixed cells (on slides) were incubated in the presence of the primary antibody at the same concentration used for cytometric analysis. After washing in PBS, the cells were labelled with a FITC-coniugated secondary antibody. Finally, the labelled cells were analysed using a confocal laser scanning microscope (TCS SP2, Leica) (Krautz-Peterson et al., 2008).
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 158–165
Antibody-labelled V. parahaemolyticus detected by FCM 161 Fig. 4. Dot plots (SSC versus FL1) and relative histograms of FITC fluorescence in different mixed bacteria samples. A. 100% V. parahaemolyticus. B. Escherichia coli ATCC 8739. C. 20% V. parahaemolyticus and 80% E. coli ATCC 8739. D. 50% V. parahaemolyticus and 50% E. coli ATCC 8739. E. 80% V. parahaemolyticus and 20% E. coli ATCC 8739.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 158–165
162 A. Manti et al. Fig. 5. Linear correlation of V. parahaemolyticus labelled with the FITC-conjugated antibody in different dilution samples by FCM.
aureus ATCC 43387 and Pseudomonas aeruginosa ATCC 9027), other environmental bacteria (S. epidermidis 102, Enterococcus faecium 311, Enterobacter cloacae 402) and some environmental Vibrio spp. isolates (V. parahaemolyticus 704, V. parahaemolyticus 604, V. alginolyticus 1513, V. alginolyticus 1407, V. fluvialis 201, V. harvey 21, V. harvey 64 and V. vulnificus 509) was analysed by FCM. As reported in Fig. 1, the antibody was found to be unreactive against all the ATCC microorganisms and some of the environmental strains tested. As regards the other Vibrio spp., the relative proportion of cells labelled was high for V. harvey 64, V. parahaemolyticus 704 and V. alginolyticus 1407 while it was slightly lower for the other Vibrio spp. analysed. These data were also confirmed by means of a direct enzyme-linked immunosorbent assay using plates coated with different Vibrio spp. isolates (V. alginolyticus 1513, V. harvey 64, V. vulnificus 509 and V. parahaemolyticus 704). The specific immunocomplexes were determined after adding horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody and the HRP substrate ABTS (Boiani et al., 2009). Our polyclonal antibody raised against V. parahaemolyticus Conero shows very low cross-reactivity against the other bacterial strains. The percentage values reach 30–25% on the V. alginolyticus 1513 and V. parahaemolyticus 704, and 19–16% on the V. harvey 64 and V. vulnificus 509, with respect to V. parahaemolyticus Conero controls (data not shown).
Antigen immunolocalization by transmission electron microscopy and confocal microscopy To demonstrate further the specificity and identify the localization of our primary polyclonal antibody, transmission electron microscope (TEM) and confocal microscope techniques were used. Figure 2 shows electron micrographs of V. parahaemolyticus Conero and E. coli ATCC 8739 cells labelled with the primary antibody and a gold-conjugated secondary antibody. Figure 3 presents confocal microscopic images of antibody-labelled V. parahaemolyticus Conero and E. coli ATCC 8739. The TEM analysis and confocal images both show a highly specific distribution of the antibody to membrane antigens of V. parahaemolyticus Conero cells (Figs 2A, B and 3A). Moreover, non-specific labelling in E. coli ATCC 8739 was once again absent in images acquired using both TEM (Fig. 2C) and confocal microscopy (Fig. 3B and C), supporting the conclusion of high specificity for our antibody for V. parahaemolyticus, as initially demonstrated by FCM. Antibody specificity in mixed bacterial suspensions Suspensions of V. parahaemolyticus Conero and E. coli ATCC 8739 were mixed at different ratios (100:0, 0:100, 20:80, 50:50 and 80:20) in order to test antibody specificity. Flow cytometry analysis of the pure V. parahaemolyticus suspension (100:0) showed a higher signal intensity (FL1)
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 158–165
Antibody-labelled V. parahaemolyticus detected by FCM 163 after labelling (Fig. 4A) compared with pure E. coli ATCC 8739 suspensions (0:100) (Fig. 4B). In suspensions containing 20% of V. parahaemolyticus Conero and 80% of E. coli ATCC 8739, a very low fluorescence signal was shown only by the labelled V. parahaemolyticus Conero cells and 80% of the cells resulted as unlabelled (Fig. 4C). In suspensions containing 50% of V. parahaemolyticus Conero and 50% of E. coli ATCC 8739 FCM, analysis showed that only 50% of the cells were labelled (Fig. 4D). Finally, in suspensions containing 80% of V. parahaemolyticus Conero and 20% of E. coli ATCC 8739, a strong fluorescence signal was shown only by the labelled V. parahaemolyticus Conero cells (Fig. 4E). Total antibodylabelled cell counts, performed in FCM by the addition of counting beads to each sample, showed good cell percentage accuracies in the mixed suspensions and thus good specificity and sensitivity. We can therefore attest that no cross-reactivity was present in the V. parahaemolyticus Conero and E. coli ATCC 8739 mixed suspensions. In order to test for linear correlation and the lowest levels of labelling that can be detected with FCM using the antibody against V. parahaemolyticus Conero, cells were seeded in artificial seawater at different dilutions ranging between 103 and 106 cells ml-1. Figure 5 shows the significant and linear correlation between Log total bacterial cell counts and Log antibody-labelled cell counts (R 2 = 0.998, P < 0.001). The detection of antibodylabelled V. parahaemolyticus Conero gave reliable counts for all dilutions, underlining the sensitivity and linearity of FCM. Similar results regarding the sensitivity and linearity of FCM have been published by another group (Chitarra et al., 2002) that investigated the detection of Xanthomonas campestris; moreover, Clarke and Pinder (1998), investigating pure suspensions of Salmonella, indicated that an accurate detection was possible by FCM down to a level of 2.2 ¥ 102 cells ml-1.
natural seawater are somehow able to inhibit the binding of the antibody to V. parahaemolyticus Conero or reduce the fluorescence signal in another more direct way. Indeed, Moreira-Turcq and Martin (1998) proposed that the presence of salts, organic and inorganic particles in seawater (that would be absent in PBS) could interfere with fluorochrome efficiency and thus result in this latter hypothesis. In this study, we also tested our antibody in natural seawater samples. First of all, an aliquot of a natural seawater sample was analysed in FCM revealing the total marine bacteria (9.5 ¥ 105 cells ml-1); another aliquot was then antibody labelled in order to analyse the presence of V. parahaemolyticus in seawater. In this experiment, the fluorescence signal was below that of the detection limit (data not shown) indicating that V. parahaemolyticus and other Vibrio species were absent in the seawater analysed. Finally, a given concentration of V. parahaemolyticus Conero (3.87 ¥ 106 cells ml-1) was inoculated into another aliquot of natural seawater; the data obtained indicate that the proportion of V. parahaemolyticus Conero labelled cells was reduced (44%) even though the specificity of the antibody was found to remain unchanged (see above). In conclusion, these data show that the primary antibody developed in our laboratory is specific for V. parahaemolyticus Conero, although a significant signal was also observed for V. parahaemolyticus 704, V. harvey 64 and V. alginolyticus 1407. Through the use of FCM techniques, V. parahaemolyticus Conero was readily detected when analysed as a pure culture, in a mixed bacteria suspension and when suspended in natural seawater. This study demonstrates the versatility of FCM for the detection of V. parahaemolyticus in seawaters and opens the way for the development of convenient test kits that could be used for V. parahaemolyticus as well as other Vibrio species.
Detection of V. parahaemolyticus Conero in artificial and natural seawater
References
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