SPECIFIC DETECTION OF VIABLE FOODBORNE PATHOGENIC BACTERIA IN FRESH-CUT VEGETABLES

Patricia Elizaquvel1, Gloria Snchez2, Natividad Saln1, Jordi Cerver1, Rosa Aznar1,2
1

Departamento de Microbiologa y Ecologa, Universitat de Valncia. Av. Dr. Moliner, 50. 46100

Burjassot. Valencia.
2

Departamento de Biotecnologa, Instituto de Agroqumica y Tecnologa de Alimentos (IATA, CSIC).

Av. Agustn Escardino, 7. 46980 Paterna. Valencia. Spain

Introduction The major challenge when applying real-time PCR (qPCR) assays for the detection of foodborne pathogens is how to distinguish between DNA from dead and live cells. This is particularly relevant for processed foods or foods subjected to long-time storage due to the relatively long persistence of DNA after cell death. A promising strategy to avoid this drawback relies on the use of DNA binding molecules like propidium monoazide (PMA) as a sample pretreatment previous to the qPCR based on the integrity of bacterial cells with compromised membranes (Nocker et al. 2008). In this study a rapid method has been developed for the concentration, detection and quantification of viable E. coli O157:H7, Salmonella and Listeria monocytogenes cells combining PMA or reagent D (commercially available reagent from Biotecon) and qPCR, in fresh-cut vegetables. Materials and methods Three reference strains supplied by the Spanish Type Culture Collection (CECT) were included in this study: Salmonella enterica ssp. enterica CECT 915T, Listeria monocytogenes CECT 4031T and Escherichia coli O157:H7 CECT 4267. They were grown in Trypticase Soy Broth (TSB) or Agar (TSA) at 37 C for 18 h. Cultures were adjusted to OD = 1 prior to inoculation. Dead cell suspensions were obtained by incubating 500 l of the broth culture with isopropanol (1:2 v/v) during 10 min at room temperature. Different concentrations of PMA (50 and 100 M) and reagent D (300 or 150 l) were tested in order to determine the optimal conditions. Treated and untreated live bacteria were diluted and plate counts were performed on TSA to determine toxicity of both reagents. In both cases, after the addition of the reagent, an incubation period of 5 min in the dark at room temperature was performed with occasional mixing to allow reagent penetration. Thereafter, samples were exposed to light for 15 min using a photoactivation system (Led-Active Blue, 1

Ingenia Biosystems). After photo-induced crosslinking, cells were centrifuged at 7000 rpm for 5 min prior to DNA isolation. Two types of fresh vegetables (spinach and salad) were used as food matrices in spiked assays to evaluate the efficiency of the PMA to distinguish between live and dead cells. Ten grams of each sample were inoculated with different concentrations of each live and isopropanol-killed pathogen, separately. After drying, 90 ml of Buffered Peptone Water (BPW) were added and sample was homogenised in a filtered sterile bag using a Pulsifier. The resulting filtrate was collected and polyethylene glycol was added to obtain a final concentration of 10%. After a gentle agitation for 1 hour at 4 C, samples were centrifuged 30 min at 10000 g. Pellets were re-suspended in 2 ml phosphate buffer saline pH 7 (PBS). DNA from both pure cultures and inoculated vegetables was purified using NucleoSpin Tissue kit (Macherey-Nagel). Detection was determined by qPCR using specific primers and probes for each pathogen (Hoorfar et al. 2000; Rodrguez-Lzaro et al. 2003;Yoshitomi et al. 2006).

Results and discussion Initially, the optimal concentration of PMA and reagent D was determined for discrimination between viable and killed bacteria (isopropanol treated) in cell suspensions (Table 1). For the three pathogens a final concentration of 50 M PMA was used, showing that the number of PMA-treated dead bacteria exhibited, as an average, a 3-4 logs decrease compared to the number of untreated dead bacteria. Reagent D showed similar reductions, in E. coli O157:H7 and Salmonella but it was toxic to L. monocytogenes and therefore was not further evaluated. PMA treatment was then assayed in two fresh-cut vegetables (salad and spinach) using different concentrations of live-dead bacteria obtained from calibrated suspensions. Results from untreated and PMA treated dead bacteria are shown in Table 2. Successful amplifications where obtained from all samples containing viable cells and most of the samples containing untreated dead bacteria, except for L. monocytogenes in salad and E. coli O157:H7 in spinach. Moreover, PMA treated live cells showed amplification levels similar to those obtained from non-treated cells (data not shown) demonstrating that PMA treatment does not affect live cells. PMA treatment inhibited qPCR detection of dead cells of the three pathogens in both matrices assayed and in all concentrations. This indicates that PMA is capable of penetrating the compromised cell membranes of dead cells.

Conclusions The evaluation of PMA and reagent D for qPCR detection of Salmonella enterica ssp. enterica, Listeria monocytogenes and Escherichia coli O157:H7 viable cells revealed that reagent D was toxic to L. monocytogenes in pure cultures. PMA at 50 M did not affect viable cells while it succeeded in penetrating compromised cell membranes of dead cells in all three pathogens. This procedure allows, in less than 8 hours, the detection and quantification of viable foodborne pathogens in fresh-cut vegetables.

References

Hoorfar,J., Ahrens,P. and Radstrm,P. (2000) Automated 5nuclease PCR assay for identification of Salmonella enterica. J. Clin. Microbiol 38, 3429-3435. Nocker,A., Cheung,C.-Y. and Camper,A.K. (2008) Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Methods 67, 310-320. Rodrguez-Lzaro,D., Hernandez,M., Scortti,M., Esteve,T., Boland-Vazquez,J.A. and Pla,M. (2003) Quantitative detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly, iap, and lin02483 :Targets and AmpliFluor Technology. Appl. Environ. Microbiol. 70, 1366-1377. Yoshitomi,K.J., Jinneman,K.C. and Weagant,S.D. (2006) Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H- using SYBR Green I in a realtime multiplex PCR. Mol. Cel. Probes 20, 31-41.

Table 1. Cp increase between dead and PMA/RD treated dead cells in pure cultures containing 108 cfu/ml. Shaded values indicate that the reagent was toxic to live cells.

E. coli O157:H7 RD 150 l 300 l PMA 50 M 100 M 15.53 16.78 16.35 7.7

L. monocytogenes 12.15 12.60 9.48 6.43

Salmonella 14.28 9.42 7.11 9.39

Table 2. Cp values obtained by specific q-PCR for each pathogen in two artificially inoculated fresh-cut vegetables. Inoculations where done in triplicate for each concentration.

Food matrix

Pathogen

Inoculation (cfu/g) 1.02 x 106 1.02 x 104

4

Cp SD Live 26.15 0.47 32.46 0.52 30.31 0.32 34.54 0.36 28.32 0.45 31.05 0.84 21.08 0.40 27.67 0.47 29.59 0.89 33.73 0.52 25.52 0.25 31.24 0.63 Dead 29.83 0.48 >35 30.28 0.70 34.77 0.31 28.70 0.74 31.14 0.30 25.67 0.56 31.15 0.37 29.99 0.38 >35 27.66 0.52 31.91 0.42 Dead PMA- treated >35 >35 >35 >35 >35 >35 >35 >35 >35 >35 >35 >35

Spinach E. coli O157:H7

L. monocytogenes

7.65 x 10

7.65 x 102 Salmonella 6.15 x 105 6.15 x 103 Salad E. coli O157:H7 1.89 x 107 1.89 x 105 L. monocytogenes 3.2 x 103 3.2 x 10 Salmonella 1.65 x 106 1.65 x 104