Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Journal of Applied Microbiology, 2004;96(5):1097-1104

Validating the use  of  green fluorescent-marked  Escherichia coli O157:H7 for assessing  the organism behaviour in foods

M. Vialette, A.-M. Jandos-Rudnik, C. Guyard, O. Legeay, A. Pinon and M. Lange

ABSTRACT

Aims: Monitoring bacterial kinetics in food is of great importance in food safety. The targeted micro-organism has to be identified accurately among competitive flora. Using green fluorescent protein (GFP)-transformed strains is a possible answer to such issues. However, quantitative studies require that this transformation does not alter the micro-organism behaviour: parent and transformed organisms were thus compared.

Methods and Results: Three Escherichia coli O157:H7 strains were transformed using a GFP-plasmid expressing. Parent and transformed strains were compared according to their genetic characteristics and serotypes. Growth ability was also assessed in constant and fluctuating temperature profiles. Cardinal values of pH, water activity and temperature were computed. No differences were observed between parent and transformed strains for all these experiments. The plasmid was satisfactorily maintained within transformed strains throughout the studies. Growth was eventually monitored in beef meat.

Conclusions: Using the GFP marker is of great value, as it allows easier enumeration of E. coli O157:H7 in food in the presence of natural microflora. Using transformed strains is legitimate: their behaviour does not differ from that of their parent strains.

Significance and Impact of the Study: GFP transformation appears to be a valuable and reliable tool for challenge testing studies and predictive microbiology.

INTRODUCTION

Although Escherichia coli is a common inhabitant of the human and animal gastrointestinal tract, several pathogenic types of the species are able to cause a variety of human diseases. First recognized as a human pathogen in 1982, E. coli O157:H7 [Shiga toxin-producing E. coli (STEC)] has since been associated with outbreaks of food-borne illness in countries across the world, with many foods identified as vehicles of infection (Bouvet et al. 2001; Fantelli and Stephan 2001; Rogerie et al. 2001). The main virulence factor of STEC is the ability to form cytotoxic exotoxins (Shiga toxins) (Boerlin et al. 1999). Two major categories of Shiga toxins have been distinguished, Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2). The other virulence factors are the property of producing attachment-effacement lesions and the presence of an entero-haemolysin gene (Law 2000).

Given the severity of the illness associated with E. coli O157:H7, it is necessary to provide the food industry with procedures for preventing its presence and controlling its growth. The aim of predictive microbiology lies in predicting growth kinetics of micro-organisms at a given moment during food processing and thus predicting the evolution of food during storage, manufacturing or preservation accidents. Rosso et al. (1993, 1995) proposed, in their cardinal approach, a model in which the maximum microbial specific growth rate ( _max) is a function of temperature or pH. The cardinal values of the considered strain are then determined. As an example, for temperature values, the following parameters are considered: T _min (the temperature below which growth is no longer observed), T _max (the temperature above which no growth occurs) and T _opt (the temperature at which the maximum specific growth rate _max equals its optimal value _opt). The same procedure can be applied to pH and water activity a _w (Augustin and Carlier 2000). The minimal and maximal values are important factors to food preservation, and the optimal value is a crucial parameter in predictive microbiology (Gonthier et al. 2001). In this approach, for a given strain, _opt is a food-dependent parameter.

The development of sensitive methods for monitoring bacteria in foods, especially in presence of natural contaminating flora, is of marked importance. Indeed, models have to be validated and it is usually more appropriate to collect new data in food to have a good validation. In this way, the objective of this work was firstly the construction of E. coli O157:H7 strains marked with the green fluorescent protein (GFP) gene carried by a plasmid. The GFP from the jellyfish Aequorea victoria, a versatile 27-kDa protein, has proved to be valuable as a tool for studying a variety of biological questions, e.g. to study gene expression, to determine protein distribution or to tag a cell lineage in vivo, in situ and in real time (Cha et al. 1999; Errampalli et al. 1999; Freitag and Jacobs 1999). The GFP emits green light when excited with ultraviolet (u.v.) radiation and no substrate or cofactor is required for fluorescence.

Ajjarapu and Shelef (1999) used GFP-bearing E. coli O157:H7 in ground beef and were able to monitor its evolution despite the presence of a background microflora. It should yet be verified that using GFP-transformed strains instead of nontransformed strains is a right way, i.e. transformed strains and parent strains should behave in a similar manner. Fratamico et al. (1997) found no differences according to morphological or biochemical criteria or using PCR. In a second part of the present work, the influence of temperature on the growth kinetics of both GFP-E. coli strains and their parental strains was assessed in liquid medium. GFP expression was evaluated during growth. Cardinal values of pH, a _w and temperature of GFP or parental strains were determined comparatively. Furthermore, experiments at varying temperature profiles were carried out in order to simulate temperature-changing conditions (food processing, food storage, etc.). The results obtained in liquid medium were complemented by experiments in food.

MATERIALS AND METHODS

Strains and media

Three E. coli O157:H7 strains were used in this study. One of the strains was isolated from bovine faeces and will be referred to as strain 1, the other two were of clinical origin and will be referred to as strains 2 and 3.

These strains were transformed with a plasmid vector pBAD-GFPuv (BD Biosciences - Clontech Inc., Palo Alto, CA, USA), adapted for visualization under u.v. light.

Stock cultures were maintained at 80°C in cryobank (AES Laboratoires, Combourg, France).

All strains were resuscitated before use by inoculation into 10 ml of brain-heart infusion (BHI) broth (Oxoid Ltd, Basingstoke, Hampshire, UK), followed by incubation at 37°C for 8 h. The subculture and culture medium was BHI (Oxoid) supplemented with yeast extract (Oxoid) 3 g l ¹ and glucose (Prolabo, Fontenay-sous-Bois, France) 2 g l ¹. The BHI supplemented medium was sterilized by filtration (0·20  m pore size). This will be referred to as modified BHI.

The count medium was plate count agar (AES Laboratoires) for the E. coli strains. Plates were incubated at 37°C for 24 h.

Construction and identification of GFP-expressing E. coli

Escherichia coli strains were transformed with pBAD-GFPuv by the calcium chloride method (Ausubel et al. 1988). The pBAD-GFPuv plasmid vector contains an arabinose-induced promoter and an ampicillin resistance gene. Transformants were selected by plating on PCA supplemented with arabinose 2 mg ml ¹ (Sigma, St Louis, MO, USA) and ampicillin 50  g ml ¹ (Aldrich Chemical Company, Milwaukee, WI, USA) (PCA + Ara + Amp). Colonies on PCA + Ara + Amp, that were induced by arabinose and ampicillin resistant, were fluorescent under u.v. light. Stability of fluorescence was monitored. Indeed, the percentage of fluorescent colonies before and after experimentations was also examined throughout the present study.

Parent and transformed strains were compared according to their genetic characteristics (stx genes), serotypes and biochemical characteristics. Detection of stx genes was performed with degenerate primers ES149 and ES151 as described by Read et al. (1992). These primers amplified a conserved sequence of stx ₁ and stx ₂ genes. For DNA preparation, 1 ml of an overnight sample broth (BHI) was centrifuged at 12 000  g for 3 min. The pellet was washed in PBS buffer (pH 7·4; Sigma). The DNA from pelleted cells was released by boiling and purified using Instagen DNA purification matrix (Bio-Rad, Ivry-sur-Seine, France). Ten microlitres of the DNA were used as template for PCR detection of stx genes. The conditions for the PCR amplification were the same as those described by Uyttendaele et al. (1998).

Serotyping of the different strains was performed for O157 and H7 determination with Difco antisera (Difco Laboratories, Osi, Maurepas, France).

Parent or transformed strains were biochemically confirmed by using the API 20E test strips (Biomérieux, Marcy l'Etoile, France).

Growth kinetics as function of constant or dynamic temperature in CARY 100

Behaviour of parent and GFP-expressing strains in function of temperature was compared in modified BHI. Parent and transformed strains were grown simultaneously in a spectrophotometer CARY 100 (Varian S.A., Les Ulis, France). Three replicates were carried out for each condition.

After resuscitation (in modified BHI, 8 h at 37°C), the parent or GFP-expressing strains were subcultured in modified BHI and in modified BHI + ampicillin (50 g ml ¹), respectively at the defined temperature until the beginning of exponential phase. A 0·5% of this inoculum, corresponding to inoculum size of 10⁶ CFU ml ¹, was then transferred to the culture medium (modified BHI) for growth assessment. Growth was monitored in constant temperature conditions at 37, 20 and 10°C. Moreover, growth evaluation was carried out in dynamic temperature conditions as follows: the strains were first placed at a start temperature until beginning of exponential phase and then, a new temperature was applied. Studied conditions were: 37°C followed by 20°C, 37°C followed by 10°C, 20°C followed by 10°C and 10°C followed by 20°C. The temperature gradient was usually achieved in <15 min.

In order to check fluorescence, plate counts (PCA + Ara) were performed at the beginning and at the end (cells in stationary phase) of every experiment.

Determination of cardinal values of pH, a _w, temperature in Bioscreen C

The methodology was defined in the framework of the French Predictive Microbiology Project, Sym'Previus as described by Membre et al. (2002). The detection time method was used to calculate _max for different conditions of temperature (tested range: 8-45°C), pH (adjusted by HCl: pH 4-7) or a _w (regulated by NaCl: 0·5-8%) in a Bioscreen C (LabSystems, Helsinki, Finland). Briefly, this method is based on the fact that successive binary dilutions produce growth curves that are shifted from each other by one-generation time value. The obtained values of _max allowed determination of cardinal values (T _min, T _opt, T _max, pH_min, pH_opt, pH_max, a _{w min}, a _{w opt}, a _{w max}) and _opt with Rosso secondary model (Rosso et al. 1995). Cardinal values a _{w max} were set to 1. Cardinal values pH_max were chosen so that pH model would be symmetric, i.e.:

Experiments in food

In order to evaluate the potential utilization of GFP strain in food product, growth of GFP-transformed strain 2 was monitored in raw ground beef at 10°C. The strain was first subcultured in modified BHI + Amp at 37°C for 8 h; a second subculture was conducted in the same medium at 10°C for 4 days. The suspension was diluted to produce an inoculation level in beef of ca 2·5  10³ CFU g ¹. Inoculated meat was divided into 10 g aliquots and placed in sterile bags.

A growth curve was produced with 10-15 points and repeated once. On each measurement time, one bag was opened for enumeration. Meat was placed in 90 ml of tryptone salt broth, and then homogenized for 1 min. Dilutions were made in tryptone salt broth to obtain appropriate levels. Plating was made on PCA for total bacterial count, on PCA + arabinose (0·2%) (PCA + Ara) for visualization of the GFP strain under u.v. light, and on PCA + arabinose (0·2%) + ampicillin (50  g ml ¹) (PCA + Ara + Amp) as a selective medium allowing expression of fluorescence.

Statistical analysis of data

Analysis of variance, regressions or confidence intervals calculations were performed using software S-Plus 2000 (AT&T Bell Laboratories, Murray Hill, NJ, USA). Graphical outputs were also produced with Microsoft Excel 2000 (Microsoft Corporation, Redmond, WA, USA).

The primary model chosen in this study was the model of Rosso (1995). It was used for constant and dynamic temperature studies and for food experiments, in order to calculate primary model parameters (mainly lag time and growth rate _max). A cardinal values model (Rosso et al. 1995) was chosen as secondary model.

RESULTS

Construction and identification of GFP-expressing E. coli

pBAD-GFPuv was successfully introduced into the three E. coli O157:H7 strains. Colonies of the transformed and parent strains had identical morphological characteristics on PCA + Ara + Amp (for GFP strains) and PCA (for parent strains), except that colonies of transformants appeared green under u.v. visualization, whereas those of the parent strains remained nonfluorescent.

The comparison of the parent and transformed strain genetic characteristics, i.e. detection of stx genes, showed that virulence genes were present after the plasmidic transformation. Furthermore, no serotypic (i.e. O157:H7) or biochemical difference was recorded between parent and transformed strains.

Stability of fluorescence was also monitored. The transformants were cultured at 37°C in the nonselective liquid medium, i.e. without ampicillin. The plasmid was maintained in more than 60% of colonies after 13 days of overnight cultures (data not shown).

Growth kinetics as function of constant or dynamic temperature

GFP-expressing strains were compared with their respective parent strains according to growth rate and lag time values (calculated from Rosso primary model) under various temperature conditions. Correct agreement between the strain results was observed. Figure 1 shows the parameter values calculated for three temperature conditions: a constant temperature condition, 20°C (Fig. 1a) and two dynamic conditions, 37 °C followed by 20°C (Fig. 1b) and 10°C followed by 20°C (Fig. 1c). The values reported in Fig. 1b,c are those obtained at 20°C, i.e. after the temperature shift. As shown by these results, no significant differences in the lag times or growth rates between the parent and GFP-expressing strains were observed, as shown by overlapping confidence intervals. The behaviour of parent and transformed strains was similar, whether the strains were placed at the defined temperature at the stationary phase (Fig. 1a) or after a temperature shift (negative or positive) at the beginning of the exponential phase (Fig. 1b,c). When considering the positive temperature variation, i.e. 10°C followed by 20°C (Fig. 1c), larger confidence intervals were observed for lag times. This is not surprising, as lag times usually exhibit a large variability under unfavourable conditions, and especially when the growth temperature changes abruptly. However, overlapping confidence intervals represent a rough indication of nondifference. A global summary of the comparison of growth rates and lag times for all tested conditions (constant or dynamic) is shown on Fig. 2. The values used for the dynamic conditions are those calculated after the temperature shift, i.e. the final temperature value. Each value of growth rate ( _max) (Fig. 2a) or lag time ( ) (Fig. 2b) for a GFP-expressing strain was plotted against the corresponding value of its parent strain, showing a general good agreement. For _max (Fig. 2a), only a few points fall outside the 95% confidence interval. As seen on Fig. 2b, more variation is observed for the lag time, but most results fall in the 95% confidence interval. Furthermore, differences were also examined using analysis of variance. The fact that a strain is expressing GFP was not found to have a significant effect on growth rate or lag time (results not shown). On the whole, the results of this study showed that the plasmidic transformation had negligible effect on the growth capacities of the E. coli strains.

Loss of GFP expression was also examined for transformed strains. Colonies plated on PCA + Ara were counted, and another count was made under u.v. light in order to evaluate the proportion of fluorescent colonies. The percentage of nonfluorescent colonies corresponded to plasmid loss within the bacterial population. As seen in Table 1, mean plasmid loss percentage was under 10% in all the cases. The maximal range of observed plasmid loss was 0-21%. These observations indicated of a correct GFP expression, and therefore a conservation of the plasmid at whatever the environmental temperature conditions. A constant temperature of 10°C gave less favourable results, but experiments were longer (5 days). Strains seemed to keep their fluorescence better in dynamic conditions (rapid temperature changes) of growth.

Cardinal values of a _w, pH, temperature

Cardinal values of a _w, pH and temperature for strain 2 are indicated in Table 2 with their confidence intervals. As shown by these results, the confidence intervals between the parent and the transformed strains are overlapping. This trend confirmed the previous observations, showing no differences between the parent strain and the GFP-expressing strain behaviour. It must be noted that the pH_max was estimated by a symmetric model and no experiment was conducted to alkaline value. This could explain the fluctuation of calculated pH_max values.

The loss of GFP-expression for transformed strain 2 was evaluated for all tested conditions of a _w, pH and temperature. The means of the plasmid loss percentage, as well as the minimal and maximal percentages, were calculated and indicated in Table 3. Whatever the environmental conditions, strain 2 exhibited a good conservation of the plasmid. In general, the plasmid loss was below 10%. This value could be more pronounced for the most severe culture conditions, e.g. in presence of the highest salt concentration, the lowest acid pH or the highest temperature. But the percentage never exceeded 33% (Table 3).

Growth in beef meat

The potentiality of use of GFP-expressing strain in food product was evaluated. A challenge-test with the transformed strain 2 was carried out in raw ground beef at 10°C. The growth kinetics are showed in Fig. 3. High counts of a competitive flora, up to 10⁵ CFU ml ¹, were initially found in meat, which represented particularly hard condition to follow the growth of a specific E. coli strain. The total flora was counted on PCA, and GFP strain on PCA + Ara and PCA + Ara + Amp (as a selective medium). As seen in Fig. 3, the evolution of the GFP strain was easily distinguished from competitive flora thanks to its fluorescence: its survival could be monitored throughout the experiment. The results obtained in the two plating media were similar.

FIGURES

Fig. 1 Mean lag times in hours (a) and growth rates in per hour (b) calculated over the three replicat...

Fig. 2 Representation of _max (a) and (b) calculated for parent strains vs GFP-expressing strains. Str...

Fig. 3 Growth curves of GFP-expressing strain 2 () counted in PCA + Ara; () counted in PCA + Ara + Amp and t...

Table 1 Mean plasmid loss percentage (over three repetitions) of GFP-Escherichia coli transformed stra...

Table 2 Cardinal values of a _w, pH and temperature of the parent and GFP strain 2

Table 3 Mean plasmid loss percentage (over three dilution levels) of GFP strain 2 at the end of the ca...

DISCUSSION

Using GFP transformation allowed for successful applications in assessing various biological issues(Bloemberg et al. 1997; Ajjarapu and Shelef 1999; Errampalli et al. 1999). In the present work, the construction and evaluation of E. coli strains carrying the GFP-expressing plasmid pBAD-GFPuv are reported.

The presence of this plasmid does not affect the intrinsic characteristics of the E. coli strains, i.e. the serotypic or biochemical traits and virulence genes. No significant behaviour difference between marked strains and parent strains could be found, as indicated by overlapping confidence intervals. A major limitation of the GFP marker is the small number of studies on the influence of environmental conditions on the GFP expression, and more particularly, for a plasmid marker (Errampalli et al. 1999). Our results have demonstrated the stability of the marker in unfavourable environment, i.e. in presence of acid, salt and in cooling/heating environment. Indeed, the study of the transformant strain cardinal values showed that the GFP persists in large ranges of pH values (4·0-7·0), NaCl concentrations (0·5-8%) and temperature (8-45°C). Furthermore, the plasmid stability was confirmed in dynamic conditions of temperature, where the extent of variation could reach 27°C. It is well known that this factor may vary extensively throughout food processing. The stress induced by the abrupt change of temperature did not increase the plasmid loss.

Most foods are complex systems with heterogeneous microbial populations. The introduction of the GFP marker provides a simple method to differentiate between inoculated strains and natural competitive flora of the product. Indeed, in the experiments on food described above, we could rapidly distinguish individual species within the natural flora of the product. GFP labelling in micro-organisms makes it a marker of choice for ecological studies. Furthermore, the stability of the plasmid in the absence of selection obviates the need for the addition of antibiotics to these systems in short-time experiments (14 days in the tested food experiment). In laboratory medium, GFP-labelled strain could be detected on the basis of green fluorescence, even after 13 days of inoculation. This observation is in accordance with the previus study of Bloemberg et al. (1997), who examined the stability of a GFP-containing plasmid in Pseudomonas spp.

Marking cells with GFP expressed from a plasmid provides the flexibility to transform a wide range of strains (Bloemberg et al. 1997; Delazari et al. 1998). Indeed, the construction of such strains is relatively easy.

Predictive food microbiology has been focusing on modelling the microbial responses to food environment, in the interest of food safety and for avoiding spoilage. Predictive models have been regularly published to describe the growth of a strain or a mixture of strains as a function of the environmental factors of food (McDonald and Sun, 1999). A good way of validating a model is to compare its prediction to real data obtained from food products (Ross et al. 2000). The standard method of data collection is the total viable count, which is a very labour-intensive method. In this way, the use of GFP-marked strain of providing a useful system for monitoring the evolution of a defined micro-organism and even developing among competitive ones, presents strong advantages.

ACKNOWLEDGEMENTS

The determination of cardinal values was realized in the framework of the French Program of Predictive Microbiology Sym'Previus, which was supported by French Ministries of Research and Agriculture. The authors would like to thank Dr Eduardo Dei-Cas (head laboratory of the Parasitology service at Pasteur Institute of Lille), for helpful and critical reading of the manuscript.

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