Fluorescent labelling negatively affects the physiology of Lactococcus lactis

International Dairy Journal 49 (2015) 130e138

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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj

Fluorescent labelling negatively affects the physiology of Lactococcus lactis
b, Henrik Max Jensen b, Gunda Hansen a, *, Claus Lindvald Johansen b, Anders Hans Honore Lene Jespersen c, Nils Arneborg c a b c

Danisco Deutschland GmbH, Busch-Johannsen-Straße 1, 25899 Niebüll, Germany DuPont Nutrition Biosciences Aps, Edwin Rahrs Vej 38, 8220 Brabrand, Denmark Department of Food Science, Food Microbiology, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 January 2015 Received in revised form 1 May 2015 Accepted 2 May 2015 Available online 19 May 2015

Culturability on plates, growth behaviour in liquid media and acidification activity of three Lactococcus lactis strains was negatively affected by carboxyfluorescein diacetate (cFDA), the corresponding succinimidyl ester cFDA-SE, propidium iodide (PI) and TOTO-1 iodide. Single staining with the vitality dye cFDA-SE decreased the reproductive capability and acidification activity of L. lactis cells more than cFDA. Since TOTO-1 proved to have a higher toxicity than PI, the application of PI was favoured over TOTO-1 as counterstain for cells labelled with one of the vitality dyes. The overall extent to which double staining with cFDA/cFDA-SE and PI impaired bacterial physiology was determined by the dye with the greater influence on L. lactis cells during single staining. The observed strain-dependent differences in sensitivity highlight the importance of studying the impact of fluorescent labelling on cell physiology before using the dyes in combination with flow cytometric cell sorting for physiological characterisation of subpopulations. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Microbial viability can be defined as the capability of cells to maintain functions required for survival, including, e.g., membrane integrity, transcription and translation, energy generation for metabolism, biosynthesis of cell components and growth (Breeuwer & Abee, 2000). Replication has often been considered as the main criterion for viability and has traditionally been measured by the ability of cells to form visible colonies on solid media (Barer & Harwood, 1999; El Arbi, Ghorbal, Delacroix-Buchet, & Bouix, 2011). A more differentiated picture of bacterial physiology is provided by analysing the metabolic activity, commonly referred to as cell vitality (Hornbæk, Dynesen, & Jakobsen, 2002). The metabolic activity of lactic acid bacteria is reflected by the acidification of milk under defined conditions and is considered as a suitable in
al, & Corrieu, 2000). dicator of starter culture quality (Fonseca, Be
al Fonseca, Marin, and Morris (2006) and Rault, Bouix, and Be (2009) applied the automated CINAC system (Corrieu, Spinnler,

* Corresponding author. Tel.: þ49 4661 602273. E-mail address: [email protected] (G. Hansen). http://dx.doi.org/10.1016/j.idairyj.2015.05.007 0958-6946/© 2015 Elsevier Ltd. All rights reserved.

Picque, & Jomier, 1988) to compare the acidification activity of Lactobacillus delbrueckii during fermentations, freezing and storage. To complement these population-averaging methods, other techniques are required facilitating the rapid differentiation of individual cells within a cell population based on their physiological state (Breeuwer & Abee, 2000; Brehm-Stecher & Johnson, 2004). Flow cytometry has evolved as one of the most powerful tools to analyse bacteria at single-cell level (Shapiro, 2000; Tracy, Gaida, & Papoutsakis, 2010). By combining flow cytometry with fluorescent labelling, multiple structural (e.g., DNA content) and functional parameters (e.g., enzyme activity, intracellular pH, membrane potential) can be detected simultaneously per cell (Shapiro, 2000). Many reviews provide an overview of various fluorescence techniques applied for viability and vitality assessment of bacteria (e.g., Breeuwer & Abee, 2000; Shapiro, 2000; Veal, Deere, Ferrari, Piper, & Attfield, 2000). The widely used vitality dye carboxyfluorescein diacetate (cFDA) gives direct proof of intracellular enzyme activity. After passive diffusion into the cell, the ester bond of the nonfluorescent precursor cFDA is hydrolysed by unspecific esterase activity resulting in the green fluorescent carboxyfluorescein (Bunthof, van den Braak, Breeuwer, Rombouts, & Abee, 1999; Riis, Pedersen, Sørensen, & Jakobsen, 1995; Rotman & Papermaster,

G. Hansen et al. / International Dairy Journal 49 (2015) 130e138

1966). The corresponding succinimidyl ester of carboxyfluorescein diacetate (cFDA-SE) binds to intracellular amines after hydrolysis and is frequently employed to determine the intracellular pH of cells (Siegumfeldt & Arneborg, 2011). Propidium iodide (PI) has been extensively used for labelling dead cells (e.g., Ananta & Knorr, 2009; Ben Amor et al., 2002). This dye is supposed to enter cells with compromised membranes and to form a red fluorescent complex with nucleic acids (Breeuwer & Abee, 2000; Bunthof et al., 1999; Johnson & Spence, 2010). TOTO-1 iodide is also described as being excluded by cells with intact membranes, but showing very high fluorescence enhancement after binding to nucleic acids (Hirons, Fawcett, & Crissman, 1994; Johnson & Spence, 2010). To simultaneously differentiate physiological states, many authors combine dyes that independently determine different parameters of microbial viability and vitality. In particular, double staining with cFDA and PI followed by flow cytometry has been reported numerous times as in situ viability assay for lactic acid bacteria and bifidobacteria (e.g., Ben Amor et al., 2002; Chen, Ferguson, Shu, & Garg, 2011; El Arbi et al., 2011; Lahtinen et al., 2006; Rault et al., 2009). Flow cytometric sorting of fluorescently labelled cells offers the possibility to physically separate subpopulations and to investigate the relationship between the labelling pattern and, e.g., the culturability of sorted cells (Ben Amor et al., 2002; Bunthof, Bloemen, Breeuwer, Rombouts, & Abee, 2001). In this context, Papadimitriou, Pratsinis, Nebe-von-Caron, Kletsas, and Tsakalidou (2007) found no negative impact of sonication and sorting on culturability of Streptococcus macedonicus ACA-DC 198 cells labelled with cFDA and PI after flow cytometric cell sorting. Bunthof et al. (2001) ascribed a deviation in recovery to stress during cell sorting of non-labelled and cFDA-labelled Lactococcus lactis NCDO 712 cells. Studying the impact of fluorescent labelling before sorting may allow fluorescent labelling to be excluded as another influencing factor during physiological characterisation of sorted subpopulations. However, few studies have investigated whether the physiology of analysed cells was affected by the applied staining method before sorting and their strategies
ard et al., differed considerably (e.g., Ben Amor et al., 2002; Monthe 2012; Ueckert, Nebe von-Caron, Bos, & ter Steeg, 1997). The variety of available information underlines the need for more comprehensive and comparable studies on the physiological consequences of fluorescent labelling. The present study aimed at elucidating whether and to what extent fluorescent labelling affects the physiology of Lactococcus lactis. For this purpose, culturability on plates, growth behaviour in liquid media and acidification activity of three L. lactis strains (S1, S2 and S3) was compared after single and double staining with cFDA, cFDA-SE, PI and TOTO-1. We demonstrate the importance of these preliminary studies to apply fluorescent labelling for flow cytometric cell sorting and downstream analysis of sorted cells. 2. Materials and methods 2.1. Bacterial strains Frozen cell material of three Lactococcus lactis strains was provided by Danisco Deutschland GmbH (Niebüll, Germany): L. lactis subsp. lactis DGCC1212 (S1), L. lactis subsp. lactis DGCC1609 (S2) and L. lactis subsp. cremoris DGCC1224 (S3) (strain number of the DuPont Global Culture Collection, DGCC). These L. lactis strains were used as model organisms for this study. After thawing, the concentrated cell suspensions were diluted with filtered Dulbecco's phosphatebuffered saline (DPBS; Life Technologies, Darmstadt, Germany) to obtain final cell concentrations of 1.2  105 ± 4.7  103 cfu mL1.

131

2.2. Fluorescent labelling The dyes 5-(and-6)-carboxyfluorescein diacetate (5,6-cFDA), the succinimidyl ester of cFDA (5,6-cFDA-SE) and propidium iodide (PI) were purchased in solid form from Life Technologies (Darmstadt, Germany). Stock solutions with 25 mM cFDA and cFDA-SE or 20 mM PI were prepared with dimethyl sulphoxide (DMSO; SigmaeAldrich Chemie GmbH, Munich, Germany). TOTO-1 iodide (Life Technologies, Darmstadt, Germany) was ordered as 1 mM solution. All stock solutions and further used working solutions (diluted 1:10 or 1:25, v/v, in DMSO) were kept in the dark at 20  C for cFDA, cFDA-SE and TOTO-1 or 5  C for PI. All samples originating from the same cell suspension of one strain were handled similarly. As a control, 1 mL of the diluted cell suspension was incubated for 15 min at 30  C. Furthermore, addition of 2 mL DMSO (final concentration 0.2%, v/v) to 1 mL of the diluted cell suspension was followed by 15 min incubation at 30  C. For single staining, 2 mL of the respective dye (stock or working solution of cFDA, cFDA-SE, PI or TOTO-1) were added to 1 mL of the diluted cell suspension. Cells were stained with cFDA or cFDA-SE (final concentrations 5 or 50 mM) for 30 min at 30  C, while PI (final concentration 1.6 or 40 mM) or TOTO-1 (final concentration 0.2 or 2 mM) were incubated for 15 min at 30  C. For double staining, PI or TOTO-1 was added after the sample had been incubated for 15 min with cFDA or cFDA-SE and the mixture was kept for further 15 min at 30  C. All samples were incubated in the dark. Low and high dye concentrations for single and double staining were tested in two separate runs; the control and a sample with DMSO were included in each run. Without any further treatment, all samples were immediately used to analyse culturability on plates, growth behaviour in liquid media and acidification activity. 2.3. Plate counting As proof of culturability, automatic plating was performed on M17 agar (Oxoid Deutschland GmbH, Wesel, Germany) with 5 g lactose L1 using the spiral plater EDDY JET (IUL Instruments €nigswinter, Germany). The control, the DMSO-treated GmbH, Ko and all fluorescently labelled cell suspensions were tenfold diluted with DPBS. The instrument spirally applied 50 mL of the cell suspension or the corresponding dilution to the surface of poured M17 agar plates in duplicate. Logarithmic mode with decreasing concentration gradient from the centre outwards was selected. After 48 h of aerobic incubation at 30  C, colonies on plates with the suitable dilution were automatically counted by a Countermat (IUL € nigswinter, Germany). Colony forming units Instruments GmbH, Ko (cfu) per mL and the percentage decrease compared with the respective control were calculated. 2.4. Growth in liquid media Growth behaviour of fluorescently labelled cells in liquid M17 media (Merck KGaA, Darmstadt, Germany) was measured as the increase of optical density (OD) in microtitre plate scale. The control, the DMSO-treated and all fluorescently labelled cell suspensions were diluted 1:100 (v/v) in liquid M17 media, corresponding to an initial cell count of approximately 103 cells mL1. Nunc Edge 96-well plates (Thermo Fisher Scientific GmbH, Dreieich, Germany) with 200 mL of each sample per well (triplicates) or pure media in the outer wells were incubated for 30 h at 30  C. The Infinite M200 PRO multimode reader (Tecan Deutschland GmbH, Crailsheim, Germany) facilitated simultaneous incubation and signal detection. OD at 660 nm was determined every 5 min after 15 s of orbital shaking.

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The four-parameter Richards model (Zwietering, Jongenburger, Rombouts, & van 't Riet, 1990) was selected as descriptive model covering the first three phases of growth curves (without death phase). In the following Equation (1) of the reparameterised Richards model is t the time (in h), N(t) the OD at time t and thus N0 the inoculation level. The asymptote A¼ln(N∞/N0) is the max OD at t¼∞, n is a dimensionless shape parameter, the max specific growth rate mm (in h1) is defined as tangent in the inflection point and the lag phase l (in h) is the x-axis intercept of this tangent.


n hm ioð1Þ 1 NðtÞ n ¼ A 1 þ n$expð1 þ nÞ$exp m $ð1 þ nÞð1þ n Þ $ðl  tÞ ln N0 A (1) The growth behaviour of treated cells in liquid media was quantitatively characterised by three growth parameters (lag phase l, max specific growth rate mm, asymptote A) derived from the modified Richards model fitted to the respective set of OD data (as ln(N(t)/N0)). The fourth parameter n was not used as a growth parameter because it only allows flexible adjustment of the inflexion point between 0 and A thereby not providing any information to compare the growth behaviour. The applied Microsoft Office Excel Solver is based on a nonlinear optimisation code and estimates values for the growth parameters by minimising the sum of square errors. 2.5. Acidification activity
pillon, France) was used to The CINAC system (AMS France, Fre analyse the acidification activity of fluorescently labelled cells. Using 1 mL of the control, of the DMSO-treated and of all fluorescently labelled cell suspensions, 100 mL of 9% (w/v) skim milk (heat treated for 30 min at 96  C) were inoculated in duplicate. Comparable with growth detection in liquid media, the initial cell concentration in milk was approximately 103 cells mL1. After continuously measuring the pH for 24 h at 30  C, three descriptors were calculated for each sample to characterise acidification kinetics in milk (Ta, time to pH 5.5, final pH after 24 h). The Ta value is the time at which the pH is 0.08 pH units under its initial value. Higher Ta and longer time to reach pH 5.5 correspond to a lower acidification activity. Moreover, the final pH after 24 h was recorded.

behaviour in liquid media and descriptors of acidification activity in milk. Besides double staining, the four dyes cFDA, cFDA-SE, PI and TOTO-1 were tested separately to assign the influence to one of the two dyes. Differences compared with the control were designated as significant based on the Dunnett's test (P < 0.05). Flow cytometric analysis of cells after single or double staining with cFDA, cFDA-SE, PI and TOTO-1 verified that all cells were labelled accordingly (increased green and/or red fluorescence compared with the unstained control) (data not shown). Fluorescent labelling protocols for viability and vitality assessment of L. lactis were adapted for the selected strains and the available instrumental setup aiming at improved fluorescence intensity and separation of the subpopulations. Lower dye concentrations or shorter incubation (e.g., 0.1, 0.5 or 1.0 mM cFDA/cFDA-SE for 5 min) did not allow adequate discrimination of the subpopulations (data not shown). Values for high dye concentrations were derived from literature to examine the effects of frequently employed concentrations on bacterial physiology.

3.1. Impact of single staining on culturability As shown in Fig. 1, single staining negatively affected culturability of L. lactis subsp. lactis S1 on solid media compared with the identically handled control. Fluorescent labelling of L. lactis subsp. lactis S1 with 5 or 50 mM cFDA significantly (P < 0.05) decreased plate counts to the same extent (averaged 15.4%; for cFDA-SE averaged 10.9%). While a decrease by 10.0 and 83.7% was detected after single staining with 1.6 or 40 mM PI, complete growth inhibition on solid media was recorded after exposure of L. lactis subsp. lactis S1 to 2 mM TOTO-1. Decrease of plate counts after single staining of L. lactis subsp. lactis S2 and L. lactis subsp. cremoris S3 with cFDA, cFDA-SE, PI or TOTO-1 proved to be dependent on the dye concentration. Compared with the other two tested L. lactis strains, the culturability of L. lactis subsp. lactis S2 was affected to a lesser extent by 2 mM TOTO-1 (Fig. 1). Addition of DMSO resulted in 2e8% decreased plate counts (Fig. 1). Considering plate counting reproducibility of 5%, deviations of more than 8% for plate counts after fluorescent labelling of L. lactis S1, S2 and S3 could be ascribed to the dyes themselves rather than to the solvent DMSO.

2.6. Statistical and principal component analysis The data for plate counts, growth parameters and acidification descriptors for three strains treated with different dye concentrations and combinations were compared using one-way analyses of variance (ANOVA). Post-hoc Dunnett's test was used to detect significant differences between each staining mean and the control mean with a 5% family error rate. Furthermore, Fisher's least significant difference (LSD) test allowed grouping strains regarding the impact of staining with an individual error rate of 5%. Statistical analyses of obtained results were performed with Minitab 16.2.4 (Minitab Inc., State College, PA, USA). Principal component analysis (PCA) of the averaged data was performed in MATLAB R2013a version 8.1.0.604, 64-bit (The MathWorks Inc., Natick, MA, USA) and in PLS Toolbox version 7.3.1 (Eigenvector Research Inc., Wenatchee, WA, USA). 3. Results The impact of fluorescent labelling on three L. lactis strains (S1, S2 and S3) was investigated by comparing culturability of stained cells on solid media, characteristic parameters for growth

3.2. Impact of single staining on growth behaviour in liquid media On average 4.7 and 10.9% longer lag phases reflected changes in growth behaviour of L. lactis subsp. lactis S1 in liquid media due to fluorescent labelling with cFDA or cFDA-SE (average of percentage deviation for 5 or 50 mM; Table 1). Besides significant (P < 0.05) lag phase elongation after single staining with 40 mM PI (28.7%) or 0.2 mM TOTO-1 (23.7%), no growth was detected for L. lactis subsp. lactis S1 cells labelled with 2 mM TOTO-1. In contrast to L. lactis subsp. lactis S1, concentration-dependent changes of lag phase duration were observed with all four dyes for L. lactis subsp. lactis S2 and L. lactis subsp. cremoris S3 (Table 1). For the three tested L. lactis strains, the lag phases were longer after single staining with TOTO-1 compared with PI. In contrast to the lag phase, none of the other two growth parameters showed significant (P > 0.05) differences compared with the respective control after single staining (except for samples where parameters were not determined as no growth was detected; Table 1).

Fig. 1. Survival of Lactococcus lactis after fluorescent labelling on solid media. Results for plate counts of stained cells on M17 stated as percentage compared with the control with 1.2  105 ± 2.1  103 cfu mL1 for Lactococcus lactis subsp. lactis S1 (a), 1.2  105 ± 3.4  103 cfu mL1 for Lactococcus lactis subsp. lactis S2 (b) and 1.3  105 ± 1.4  103 cfu mL1 for Lactococcus lactis subsp. cremoris S3 (c). Values are means ± SD of duplicate experiments with the three strains for the percentage deviations compared with the control; significant (P < 0.05) differences compared with the control are indicated by an asterisk. As control, the diluted cell suspension was incubated for 15 min at 30  C; treatments were the addition of dimethyl sulphoxide (DMSO), carboxyfluorescein diacetate (cFDA), carboxyfluorescein diacetate succinimidyl ester (cFDA-SE), propidium iodide (PI) and/or TOTO-1 iodide (TOTO1). Two dye concentrations are compared: low (1) comprising 5 mM cFDA, 5 mM cFDA-SE, 1.6 mM PI, 0.2 mM TOTO-1 and high (2) comprising 50 mM cFDA, 50 mM cFDA-SE, 40 mM PI, 2 mM TOTO-1.

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Table 1 Estimated parameters (lag phase l, max specific growth rate mm, asymptote A) derived from the modified Richards model characterising the growth behaviour of Lactococcus lactis S1, S2 and S3 after fluorescent labelling in 200 mL M17 media as optical density (OD) at 660 nm.a Strain and treatment

Impact of low dye concentration

l (h) Lactococcus lactis subsp. lactis S1 Control 12.28 ± 0.01 DMSO 12.32 ± 0.13 cFDA 12.83 ± 0.08 cFDA-SE 13.50 ± 0.12* PI 12.95 ± 0.08 TOTO-1 15.19 ± 0.32* cFDA and PI 13.30 ± 0.03* cFDA-SE and PI 13.84 ± 0.21* cFDA and TOTO-1 15.93 ± 0.12* cFDA-SE and TOTO-1 19.71 ± 1.10* Lactococcus lactis subsp. lactis S2 Control 16.15 ± 0.31 DMSO 16.27 ± 0.09 cFDA 16.59 ± 0.31 cFDA-SE 16.83 ± 0.37* PI 16.38 ± 0.33 TOTO-1 17.18 ± 0.22 cFDA and PI 16.71 ± 0.37* cFDA-SE and PI 17.05 ± 0.09* cFDA and TOTO-1 17.90 ± 0.24* cFDA-SE and TOTO-1 18.27 ± 0.08* Lactococcus lactis subsp. cremoris S3 Control 9.68 ± 0.15 DMSO 9.74 ± 0.03 cFDA 9.76 ± 0.10 cFDA-SE 9.90 ± 0.03 PI 9.77 ± 0.07 TOTO-1 10.75 ± 0.06* cFDA and PI 10.05 ± 0.06* cFDA-SE and PI 10.03 ± 0.21 cFDA and TOTO-1 10.70 ± 0.10* cFDA-SE and TOTO-1 12.02 ± 0.34*

Impact of high dye concentration

mm (h1)

mm (h1)

l (h)

A

0.37 0.37 0.38 0.38 0.37 0.37 0.38 0.38 0.39 0.39

± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.03 0.03 0.03 0.02 0.02 0.02 0.03 0.07

1.99 2.02 2.01 1.99 1.96 1.96 1.98 1.99 2.02 1.72

± ± ± ± ± ± ± ± ± ±

0.04 0.03 0.03 0.06 0.07 0.04 0.04 0.06 0.05 0.21*

11.78 11.93 12.35 13.18 15.15 ND 13.16 13.95 ND ND

± ± ± ± ±

0.56 0.50 0.53 0.56 0.54 0.53 0.54 0.57 0.63 0.54

± ± ± ± ± ± ± ± ± ±

0.02 0.03 0.08 0.04 0.05 0.01 0.05 0.02 0.03 0.03

2.04 2.01 2.09 2.09 2.09 2.06 2.08 2.09 2.08 2.07

± ± ± ± ± ± ± ± ± ±

0.06 0.12 0.04 0.02 0.01 0.02 0.02 0.01 0.02 0.03

15.70 16.04 16.58 17.32 16.51 18.95 16.97 17.02 18.98 21.95

± ± ± ± ± ± ± ± ± ±

0.66 0.64 0.67 0.65 0.67 0.69 0.70 0.64 0.65 0.65

± ± ± ± ± ± ± ± ± ±

0.07 0.04 0.03 0.07 0.02 0.02 0.07 0.04 0.06 0.07

2.12 2.05 2.10 2.11 2.10 2.10 2.10 2.10 2.10 2.10

± ± ± ± ± ± ± ± ± ±

0.02 0.09 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.02

A

0.37 0.38 0.37 0.37 0.39 ND 0.37 0.39 ND ND

± ± ± ± ±

0.21 0.11 0.21 0.26* 0.90 0.50* 0.17* 0.41* 0.40* 0.56*

0.56 0.55 0.58 0.53 0.57 0.57 0.58 0.58 0.51 0.54

± ± ± ± ± ± ± ± ± ±

0.05 0.04 0.05 0.01 0.11 0.05 0.03 0.06 0.01 0.03

2.10 2.10 2.10 2.10 2.08 2.09 2.10 2.09 2.09 2.06

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.02 0.03 0.03

9.23 ± 0.03 9.21 ± 0.06 11.57 ± 0.35* 10.00 ± 0.04* 9.99 ± 0.13* ND 10.63 ± 0.10* 10.13 ± 0.14* ND ND

0.66 0.70 0.65 0.66 0.69 ND 0.70 0.65 ND ND

± ± ± ± ±

0.03 0.04 0.02 0.07 0.07

2.10 2.10 2.11 2.11 2.10 ND 2.10 2.10 ND ND

± ± ± ± ±

0.01 0.01 0.01 0.01 0.01

0.22 0.13 0.02* 0.23* 0.51*

± 0.29* ± 0.03*

0.01 0.01 0.01 0.01 0.03

± 0.01 ± 0.01

± 0.03 ± 0.08

1.97 2.00 2.04 2.01 1.98 ND 1.99 1.96 ND ND

± ± ± ± ±

0.03 0.02 0.14 0.06 0.06

± 0.05 ± 0.01

± 0.01 ± 0.01

a Values are means ± SD and are based on triplicate experiments for the three strains; an asterisk indicates significant (P < 0.05) difference compared with the control, i.e., the diluted cell suspension incubated for 15 min at 30  C. Treatments were the addition of dimethyl sulphoxide (DMSO), carboxyfluorescein diacetate (cFDA), carboxyfluorescein diacetate succinimidyl ester (cFDA-SE), propidium iodide (PI) and/or TOTO-1 iodide (TOTO-1). Low dye concentrations were 5 mM cFDA, 5 mM cFDA-SE, 1.6 mM PI, 0.2 mM TOTO-1; high dye concentrations were 50 mM cFDA, 50 mM cFDA-SE, 40 mM PI, 2 mM TOTO-1. ND indicates that no growth was detectable.

3.3. Impact of single staining on acidification activity Increased Ta and time to pH 5.5 demonstrated the negative impact of fluorescent labelling on the acidification activity of L. lactis subsp. lactis S1 compared with the respective control (Table 2). In contrast to cFDA, single staining with 5 or 50 mM cFDA-SE resulted in significant (P < 0.05) differences with a 6.3 and 9.4% higher Ta. The effect of TOTO-1 on the acidification activity of L. lactis subsp. lactis S1 was again proven to depend on the dye concentration and was higher than for PI: compared with the respective control 18.4% higher Ta for 0.2 mM TOTO-1, 78.1% for 2 mM TOTO-1. The acidification activity of all three L. lactis strains was affected to a higher extent by cFDA-SE than by cFDA (Table 2). Although single staining of L. lactis subsp. lactis S2 and L. lactis subsp. cremoris S3 with PI and TOTO-1 showed the same trends as observed for L. lactis subsp. lactis S1, the overall impact was lower. While variations due to fluorescent labelling were reflected by the Ta and the time to pH 5.5, final pH after 24 h did not vary significantly (P > 0.05) for the three tested L. lactis strains after single staining (except for L. lactis subsp. lactis S1 after TOTO-1 staining; Table 2). 3.4. Impact of double staining on culturability All tested dye combinations significantly (P < 0.05) affected the culturability of L. lactis subsp. lactis S1 on solid media (Fig. 1). Fluorescent labelling with low or high concentrations of cFDA and PI led to 23.1 and 73.7% lower plate counts; this was comparable

with when cFDA was replaced by cFDA-SE. While double staining of L. lactis subsp. lactis S1 with 5 mM cFDA/cFDA-SE and 0.2 mM TOTO-1 decreased plate counts by 76.4 and 88.9%, higher dye concentrations caused complete growth inhibition on solid media. After double staining with cFDA/cFDA-SE and PI, similar effects but to a lesser extent were detected for culturability of L. lactis subsp. lactis S2 and L. lactis subsp. cremoris S3 (Fig. 1). By using cFDA/cFDASE in combination with TOTO-1, L. lactis subsp. lactis S2 was affected to a lower degree compared with the other two tested L. lactis strains. 3.5. Impact of double staining on growth behaviour in liquid media As shown in Table 1, double staining of L. lactis subsp. lactis S1 with cFDA/cFDA-SE and PI resulted in significantly (P < 0.05) longer lag phase durations compared with the respective control (8.3 and 12.7% for low dye concentrations, 11.8 and 18.5% for high dye concentrations). With a 60.5% longer lag phase, double staining of L. lactis subsp. lactis S1 with 5 mM cFDA-SE and 0.2 mM TOTO-1 affected growth behaviour in liquid media to a higher extent than 5 mM cFDA and 0.2 mM TOTO-1 (29.8%). The concentration-dependent impact of cFDA/cFDA-SE in combination with PI on lag phase duration was also confirmed for L. lactis subsp. lactis S2 and L. lactis subsp. cremoris S3 (Table 1). In contrast to L. lactis subsp. lactis S2, growth in liquid media of the other two strains was entirely inhibited by double staining with 50 mM cFDA/cFDA-SE and 2 mM TOTO-1. Except for L. lactis subsp. lactis S1 stained with 5 mM cFDA and 0.2 mM TOTO-1, double staining did not result in significant

G. Hansen et al. / International Dairy Journal 49 (2015) 130e138

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Table 2 Influence of fluorescent labelling on the acidification activity of Lactococcus lactis S1, S2 and S3 in milk.a Strain and treatment

Impact of low dye concentration Ta (h)

Lactococcus lactis subsp. lactis S1 Control 10.67 ± 0.09 DMSO 10.73 ± 0.01 cFDA 10.93 ± 0.09 cFDA-SE 11.33 ± 0.01* PI 10.67 ± 0.01 TOTO-1 12.63 ± 0.33* cFDA and PI 10.87 ± 0.01 cFDA-SE and PI 11.33 ± 0.09* cFDA and TOTO-1 13.63 ± 0.24* cFDA-SE and TOTO-1 14.73 ± 0.19* Lactococcus lactis subsp. lactis S2 Control 10.37 ± 0.14 DMSO 10.43 ± 0.14 cFDA 10.33 ± 0.01 cFDA-SE 11.20 ± 0.09* PI 10.60 ± 0.09 TOTO-1 10.70 ± 0.24 cFDA and PI 10.73 ± 0.01 cFDA-SE and PI 11.30 ± 0.14* cFDA and TOTO-1 11.30 ± 0.14* cFDA-SE and TOTO-1 11.53 ± 0.09* Lactococcus lactis subsp. cremoris S3 Control 8.93 ± 0.01 DMSO 8.93 ± 0.09 cFDA 9.03 ± 0.05 cFDA-SE 9.30 ± 0.05* PI 9.07 ± 0.09 TOTO-1 9.60 ± 0.01* cFDA and PI 9.03 ± 0.05 cFDA-SE and PI 9.17 ± 0.14 cFDA and TOTO-1 9.97 ± 0.05* cFDA-SE and TOTO-1 11.03 ± 0.24*

Time to pH 5.5 (h)

Impact of high dye concentration Final pH after 24 h

Ta (h)

Time to pH 5.5 (h)

Final pH after 24 h

14.60 14.67 14.87 15.23 14.60 16.97 14.97 15.43 17.70 19.00

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.09* 0.05* 0.01 0.14* 0.05* 0.14* 0.05* 0.01*

4.26 4.25 4.26 4.26 4.27 4.38 4.29 4.27 4.38 4.39

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.02* 0.01 0.03 0.02* 0.01*

10.33 10.37 10.73 11.30 11.47 18.40 11.70 12.00 17.37 19.87

± ± ± ± ± ± ± ± ± ±

0.01 0.14 0.09 0.05* 0.09* 0.01* 0.05* 0.19* 0.33* 0.01*

14.30 14.40 14.73 15.27 15.50 22.67 15.80 16.17 21.50 24.07

± ± ± ± ± ± ± ± ± ±

0.05 0.09 0.01* 0.01* 0.05* 0.01* 0.01* 0.05* 0.24* 0.01*

4.28 4.28 4.27 4.29 4.29 4.79 4.37 4.32 4.55 5.42

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01* 0.01* 0.01* 0.03* 0.01*

14.33 14.40 14.30 15.10 14.50 14.63 14.70 15.30 15.30 15.60

± ± ± ± ± ± ± ± ± ±

0.09 0.01 0.05 0.14* 0.05 0.05* 0.05* 0.05* 0.05* 0.01*

4.26 4.27 4.26 4.27 4.27 4.29 4.25 4.28 4.29 4.30

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.04 0.02 0.01 0.01 0.02

9.83 9.70 10.80 10.80 10.23 10.93 11.33 11.10 11.80 12.43

± ± ± ± ± ± ± ± ± ±

0.05 0.05 0.01* 0.09* 0.05* 0.01* 0.09* 0.14* 0.09* 0.14*

13.67 13.63 14.83 14.67 14.20 15.00 15.10 15.20 15.93 16.60

± ± ± ± ± ± ± ± ± ±

0.09 0.05 0.05* 0.01* 0.01* 0.01* 0.05* 0.19* 0.01* 0.01*

4.25 4.25 4.27 4.26 4.25 4.26 4.32 4.28 4.29 4.31

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.02 0.02 0.01 0.01* 0.01* 0.01* 0.01*

12.67 12.60 12.63 12.87 12.60 13.13 12.73 13.03 13.53 14.67

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.05 0.01* 0.01 0.01* 0.01 0.05* 0.01* 0.01*

4.23 4.22 4.22 4.23 4.23 4.25 4.23 4.21 4.27 4.25

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.07

9.00 8.97 9.27 9.37 9.17 10.10 9.20 9.47 11.03 17.47

± ± ± ± ± ± ± ± ± ±

0.01 0.05 0.01 0.14* 0.14 0.05* 0.01 0.19* 0.14* 0.01*

12.70 12.63 12.83 13.03 12.90 13.80 12.87 13.20 14.63 21.33

± ± ± ± ± ± ± ± ± ±

0.05 0.05 0.05 0.05* 0.05 0.01* 0.01 0.09* 0.14* 0.01*

4.24 4.24 4.25 4.24 4.22 4.25 4.26 4.24 4.26 4.52

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.03 0.01 0.01 0.01 0.01 0.01 0.01*

a Ta is the time at which the pH was 0.08 pH units under its initial value. Values are means ± SD and are based on duplicate experiments for the three strains; an asterisk indicates significant (P < 0.05) difference compared with the control, i.e., the diluted cell suspension incubated for 15 min at 30  C. Treatments were the addition of dimethyl sulphoxide (DMSO), carboxyfluorescein diacetate (cFDA), carboxyfluorescein diacetate succinimidyl ester (cFDA-SE), propidium iodide (PI) and/or TOTO-1 iodide (TOTO-1). Low dye concentrations were 5 mM cFDA, 5 mM cFDA-SE, 1.6 mM PI, 0.2 mM TOTO-1; high dye concentrations were 50 mM cFDA, 50 mM cFDA-SE, 40 mM PI, 2 mM TOTO-1.

(P > 0.05) changes of max specific growth rate mm or asymptote A¼ln(N∞/N0) for the tested L. lactis strains (Table 1). 3.6. Impact of double staining on acidification activity Acidification activity of L. lactis subsp. lactis S1 was more affected by higher concentrations of cFDA/cFDA-SE and PI (e.g., 6.3% higher Ta for 5 mM cFDA-SE and 1.6 mM PI, 16.1% for 50 mM cFDA-SE and 40 mM PI; Table 2). Double staining with cFDA and TOTO-1 resulted in significantly (P < 0.05) higher Ta (27.8 and 68.1% for the two dye concentrations), but replacing cFDA with cFDA-SE further diminished the acidification activity of L. lactis subsp. lactis S1 (38.1 and 92.3% higher Ta for the two dye concentrations). Similar trends for the acidification activity of the three tested L. lactis strains were detected after double staining with cFDA/cFDASE and PI (Table 2). Compared with L. lactis subsp. lactis S1 and L. lactis subsp. cremoris S3, Ta and time to pH 5.5 of L. lactis subsp. lactis S2 were affected to a lower degree using cFDA/cFDA-SE in combination with TOTO-1. The final pH after 24 h was significantly (P < 0.05) higher after double staining of L. lactis subsp. lactis S1 and S2 with high concentrations of cFDA/cFDA-SE in combination with PI or TOTO-1. 3.7. Comparison of the staining impact on different L. lactis strains and detection of correlations between physiological parameters by PCA Besides comparing the impact of staining with the respective control for each L. lactis strain, the three strains were grouped based on Fisher's LSD test according to the percentage of deviation after

each treatment compared with the control (data not shown). Many grouping results assign L. lactis subsp. lactis S2 and L. lactis subsp. cremoris S3 to the same group, especially regarding the impact of fluorescent labelling on growth behaviour in liquid media. As to the impact of 2 mM TOTO-1 in single and double staining approaches, L. lactis subsp. lactis S1 and L. lactis subsp. cremoris S3 form one group with similar deviations for the various parameters. PCA was applied to investigate relationships between treatments and physiological parameters as well as correlations among the physiological parameters. High concentrations of TOTO-1 in single and double staining resulted in no growth on solid and in liquid media for L. lactis S1 and S3. These samples were found to be outliers and were excluded for further PCA. Fig. 2 shows the scores and loadings for the first and second principal component (PC) of the PCA for the averaged data after autoscaling and outlier removal. In the score plot, the three tested L. lactis strains were separated into individual groups and for each strain a clustering of the respective control with some treatments was detected. Several extremes deviated from this cluster and displayed a trend towards decreasing viability in direction of PC1. The relative shift of all treatments compared with the control was larger for L. lactis subsp. lactis S1 than for the other two strains. For all tested L. lactis strains, higher impact of fluorescent labelling was positively correlated with increasing lag phase and decreasing acidification activity and negatively correlated with culturability. According to the loading plot, the three acidification descriptors were closely clustered and in a similar way the max specific growth rate and the asymptote provided comparable information. These two growth parameters

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a

b

Fig. 2. PCA score and loading scatterplot for averaged and pre-processed data (autoscaling and outlier removal) including culturability on solid media, characteristic parameters for growth behaviour in liquid media and descriptors of acidification activity in milk of three Lactococcus. lactis strains (S1, S2 and S3) after fluorescent labelling. Panel a shows scores of PC2 versus PC1 for Lactococcus lactis subsp. lactis S1 (;), Lactococcus lactis subsp. lactis S2 (✴) and Lactococcus lactis subsp. cremoris S3 (-) with the 95% confidence interval; extremes are labelled with dye(s) and corresponding concentration level (1 for low and 2 for high dye concentrations). Panel b shows loadings for culturability (A), growth parameters (:) and acidification descriptors (þ) of PC2 versus PC1.

and the culturability on plates (as log plate counts) were oppositely correlated in their loadings for PC1 to lag phase and the three acidification descriptors. 4. Discussion Many authors have reported using cFDA or the corresponding succinimidyl ester cFDA-SE to assess the vitality of lactic acid bacteria and bifidobacteria (Ben Amor et al., 2002; Bunthof et al., 2001; Lahtinen et al., 2006). For cFDA-SE, Ueckert et al. (1997) stated no toxic effects of up to 359 mM on Lactobacillus plantarum LA 11-10 since lag times and growth yields did not vary significantly. However, the vitality dyes have never been compared with respect to their physiological impact on cells. In our study, culturability on solid media, growth behaviour in liquid media and acidification activity in milk of L. lactis S1, S2 and S3 were negatively affected after cFDA staining. Moreover, almost all results for plate counts, growth parameters in liquid media and acidification descriptors indicate a stronger and concentration-dependent influence of cFDA-SE staining (using 5 and 50 mM) compared with cFDA on the three tested L. lactis strains. This observation might be related to the fluorescent product of cFDA-SE binding to intracellular and cellsurface proteins (Johnson & Spence, 2010), a hypothesis that needs further investigations. Ben Amor et al. (2002) classified 7.5 mM PI as being non-toxic after detecting growth for electroporated PI- and subsequently cFDA-stained Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 cells. To our knowledge, the impact of using only PI on lactococci has not been reported elsewhere. We observed decreased culturability of the tested L. lactis strains after single staining with 40 mM PI demonstrating a negative impact of PI in higher concentrations on bacterial physiology. Davey and Hexley (2011) detected PI-labelled cells immediately after exposure of Saccharomyces cerevisiae to stress, but short incubation allowed the membrane to be repaired. This transient membrane permeability could explain how PI entered and negatively affected the reproductive capability and acidification activity of cells that were supposed to be viable in our study.

Fluorescent labelling with TOTO-1 was applied for quantification of Escherichia coli by Guindulain, Comas, and Vives-Rego (1997) and of lactic acid bacteria by Bunthof et al. (2001). However, our results clearly show that culturability on solid media, growth behaviour in liquid media and acidification activity of L. lactis S1, S2 and S3 were negatively affected using 0.2 mM TOTO-1. Moreover, 2 mM TOTO-1 completely inhibited growth of L. lactis subsp. lactis S1 and L. lactis subsp. cremoris S3 on solid and in liquid media. Compared with PI, TOTO-1 had a stronger negative effect on the three tested strains of L. lactis. This higher toxicity might be ascribed to the enhanced affinity of TOTO-1 with four positive charges to nucleic acids (Johnson & Spence, 2010). However, the underlying mechanisms have to be elucidated. Shen, Bos, and Brul (2009) proposed that 11 mM cFDA and 7.5 mM
ard PI did not affect culturability of Bacillus subtilis PS832. Monthe et al. (2012) found variations in side scatter of Candida shehatae cells after fluorescent labelling with 5.4 mM cFDA and 1.5 mM PI; they ascribed it to toxic compounds such as acetone and DMSO in the staining solutions rather than to the dyes. However, in our study double staining with 5 or 50 mM cFDA and 1.6 or 40 mM PI was shown to have an influence on the tested L. lactis strains which was higher than for DMSO alone. After double staining of L. lactis cells with high concentrations of cFDA/cFDA-SE and PI, the results for plate counts corresponded to single staining with 40 mM PI. The deviations for lag phase and the two acidification descriptors of L. lactis cells stained with cFDA or cFDA-SE and PI mostly reflected the negative impact observed after single staining with cFDA or cFDA-SE. Therefore, neither additive nor synergistic effects of double staining with these dyes were observed. Instead, the dye with the higher impact in single staining seemed to determine the overall extent of impairment as consequence of double staining. Bunthof et al. (2001) favoured double staining using cFDA and TOTO-1 over cFDA and PI for in situ viability assessment of different lactic acid bacteria as it facilitates the differentiation of subpopulations. However, our results for three L. lactis strains showed that cFDA together with TOTO-1 resulted in decreased reproductive capability and acidification activity compared with double staining

G. Hansen et al. / International Dairy Journal 49 (2015) 130e138

with cFDA and PI. Changes of the three analysed physiological parameters confirmed the higher impact of TOTO-1 even in combination with cFDA-SE. Hence, applying PI as counterstain for cells labelled with one of the vitality dyes was superior to TOTO-1 in our study. With regard to the negative impact of cFDA-SE and TOTO-1 separately, double staining with these dyes resulted in further decreased plate counts, longer lag phases and lower acidification activity of L. lactis S1, S2 and S3. This indicates a synergistic effect of cFDA-SE and TOTO-1 staining that was not observed for the other dye combinations. The three L. lactis strains included in this study proved to be affected by single and double staining, but the sensitivity towards florescent labelling varied between them. By taking all treatments in consideration, PCA indicated L. lactis subsp. lactis S1 to be more susceptible to fluorescent labelling than the other two strains. The PCA and the grouping results for the two L. lactis subsp. lactis strains and the L. lactis subsp. cremoris strain showed that differences were €uber and Müller (2010) not related to the subspecies level. Stra highlighted the diversity of bacteria that could cause differences in uptake and transformation of fluorescent dyes. All procedures were adjusted for flow cytometric cell sorting of fluorescently labelled L. lactis cells and subsequent physiological characterisation of sorted cells. Therefore, the extent of the fluorescent labelling effects reflected the influence that would have to be expected for similar experimental setups. Although some of the statistically significant effects were quite small, PCA verified them as physiologically relevant and associated particularly TOTO-1 treatments with an increased negative impact on L. lactis physiology. Moreover, PCA confirmed that plate counts, growth parameters in liquid media and acidification descriptors were negatively affected by fluorescent labelling as compared with the controls. All three groups of physiological parameters proved to contribute to the overall picture of the impact on the L. lactis strains. For the three L. lactis strains tested in our study, fluorescent labelling with 5 mM cFDA as vitality dye with or without 1.6 mM PI as counterstain affected bacterial physiology to the lowest extent. As consequence of the here observed strain-dependent effects of fluorescent labelling, other studies should include comparable investigations to find labelling procedures with minimal impact on the physiological state of their cells.

5. Conclusions Taken together, the results of this study demonstrated that culturability on solid media, growth behaviour in liquid media and acidification activity of three L. lactis strains were negatively affected by fluorescent labelling with all dyes tested. We found the effects of cFDA, cFDA-SE, PI and TOTO-1 to be concentration- and strain-dependent. Comparing the two vitality dyes, cFDA-SE proved to have a stronger influence than cFDA on all three investigated L. lactis strains. Single staining with either PI or TOTO-1 dereased the reproductive capability and acidification activity of L. lactis cells, but a higher toxicity was detected for TOTO-1. Combining cFDA or cFDA-SE with PI or TOTO-1 negatively affected the physiology of the L. lactis strains included in this study. The observed changes indicate that the dye with the higher impact in single staining determines the overall staining impact. In line with the results for single staining, TOTO-1 together with one of the vitality dyes was found to cause greater decreases compared with PI in double staining approaches. Referring to single staining using either cFDA-SE or TOTO-1, the combination of these dyes further impaired culturability, growth behaviour and acidification activity thereby showing a synergistic effect of double staining.

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