specific detection of viable foodborne pathogen

7/27/2019 Specific Detection of Viable Foodborne Pathogen

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SPECIFIC DETECTION OF VIABLE FOODBORNE PATHOGENIC BACTERIA IN

FRESH-CUT VEGETABLES

Patricia Elizaquvel1

, Gloria Snchez2

, Natividad Saln1

, Jordi Cerver1

, Rosa Aznar1,2

1Departamento de Microbiologa y Ecologa, Universitat de Valncia. Av. Dr. Moliner, 50. 46100

Burjassot. Valencia.

2Departamento 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 andListeria 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 andEscherichia 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,


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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. Tengrams 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 andSalmonella but it was toxic toL. 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 forL. monocytogenes in salad andE. 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.


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Conclusions

The evaluation of PMA and reagent D for qPCR detection of Salmonella enterica ssp.

enterica,Listeria monocytogenes andEscherichia coli O157:H7 viable cells revealed thatreagent 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 ofSalmonella 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 ofListeria monocytogenes andListeria innocua by

Real-Time PCR: Assessment ofhly, iap, andlin02483 :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 ofE. coli O157:H7/H- using SYBR Green I in a real-

time multiplex PCR.Mol. Cel. Probes 20, 31-41.


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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 L. monocytogenes Salmonella

150 l 15.53 12.15 14.28RD

300 l 16.78 12.60 9.42

50 M 16.35 9.48 7.11PMA

100 M 7.7 6.43 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.

Cp SDFood

matrixPathogen

Inoculation

(cfu/g) Live DeadDead

PMA- treated

1.02 x 106 26.15 0.47 29.83 0.48 >35E. coli O157:H7

1.02 x 104

32.46 0.52 >35 >35

7.65 x 104 30.31 0.32 30.28 0.70 >35L. monocytogenes

7.65 x 102 34.54 0.36 34.77 0.31 >35

6.15 x 105 28.32 0.45 28.70 0.74 >35

Spinach

Salmonella

6.15 x 103 31.05 0.84 31.14 0.30 >35

1.89 x 107 21.08 0.40 25.67 0.56 >35E. coli O157:H7

1.89 x 105 27.67 0.47 31.15 0.37 >35

3.2 x 103

29.59 0.89 29.99 0.38 >35L. monocytogenes3.2 x 10 33.73 0.52 >35 >35

1.65 x 106 25.52 0.25 27.66 0.52 >35

Salad

Salmonella

1.65 x 104 31.24 0.63 31.91 0.42 >35

specific detection of viable foodborne pathogen

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