obiology 111 (2006) 21–25www.elsevier.com/locate/ijfoodmicro

International Journal of Food Micr

Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenesand Salmonella typhimurium in artificially inoculated wheat grain☆

Jongsoo Kim, Tigst Demeke ⁎, Randy M. Clear, Susan K. Patrick

Canadian Grain Commission, Grain Research Laboratory, Winnipeg, Manitoba, Canada R3C 3G8

Received 9 September 2005; received in revised form 1 February 2006; accepted 25 April 2006

Abstract

A multiplex PCR procedure was established to detect Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificiallyinoculated wheat grain. The PCR protocol with an enrichment step successfully detected all three organisms inoculated together in non-autoclavedwheat grain. After a one day enrichment, E. coli, L. monocytogenes and S. typhimurium were detected at levels of 56, 1800 and b54 CFU/mL,respectively, in the initial sample. For L. monocytogenes, an improved detection limit of b62 CFU/mL was achieved using singleplex PCR. Forautoclaved wheat grain inoculated with the three bacterial strains individually, a detection limit of 3 CFU/mL was achieved after an enrichmentstep. The ability to test for the three bacteria simultaneously will save time and increase the ability to assure grain quality.© 2006 Elsevier B.V. All rights reserved.

Keywords: Multiplex PCR; E. coli; S. typhimurium; L. monocytogenes; Enrichment; Limit of detection

1. Introduction

The microbiological safety of food is a significant concern ofconsumers and industries today. The rapid and accurate identi-fication of bacterial pathogens in foods is important, both forquality assurance and to trace bacterial pathogens within thefood supply (Bhagwat, 2003). Grain is considered to be aproduct with a low risk of contamination with pathogenic bac-teria due to its low water activity (Berghofer et al., 2003).Although grain storage practices are not conducive to growth ofbacteria, several studies have indicated the presence of lowlevels of Escherichia coli, Salmonella spp., Bacillus cereus andvarious food spoilage microorganisms in wheat and flour due toboth pre- and post-harvest contamination (Eyles et al., 1989;Richter et al., 1993; Berghofer et al., 2003). The microbiolog-ical quality of the grain is considered to have an impact on thequality of the end product (Berghofer et al., 2003), and manyprocessors monitor the microbial load of the raw grain. Thesampling protocols employed and the extent of the information

☆ Contribution No. 917 from the Grain Research Laboratory of the CanadianGrain Commission.⁎ Corresponding author. Tel.: +1 204 984 4582; fax: +1 204 983 0724.E-mail address: tdemeke@grainscanada.gc.ca (T. Demeke).

0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2006.04.032

sought vary between companies and are not typically in thepublic domain. Buyers of grain can place the types and numbersof microorganisms into a contract specification, which thenrequires that the shipment in question be tested for those or-ganisms. Interest in the microbial load of a product may take theform of an inquiry into the historic record, from which one canprepare a statement of assurance that does not require testing ofa particular shipment. Customer standards for acceptable levelsof contamination are variable, and may be needless or ill-ad-vised (International Commission on Microbiological Specifica-tions for Foods, 1986). Inquiries from buyers and processors arenot always based on a knowledge of science, and can encom-pass organisms known to be absent from grain to specifications,such as free from bacteria and moulds, that are impossible tomeet. There are recommended tolerances for some pathogens ingrain, although the emphasis is typically on the finished product(International Commission on Microbiological Specificationsfor Foods, 1986). Grain sellers who know which organisms arepresent in their product, and their frequency, are better able torespond quickly and effectively to the inquiries and concerns ofthe grain trade.

Salmonella strains can cause general infection, food poison-ing and Salmonellosis, a zoonotic disease of considerable im-portance (Davies and Hinton, 2000). Although E. coli is the

22 J. Kim et al. / International Journal of Food Microbiology 111 (2006) 21–25

predominant facultative anaerobe of the human colonic flora,some strains are responsible for enteric disease (Abd-El-Haleemet al., 2003; Bischoff et al., 2005). Major disease outbreaks andnumerous sporadic cases of listeriosis occurring world-widehave implicated Listeria monocytogenes as another major foodborne pathogen. Although L. monocytogenes has been isolatedfrom a variety of foods (Norrung et al., 1999; Inoue et al., 2000;Rocourt et al., 2000; Maijala et al., 2001), it has not been foundon grain. However, its importance in human disease has resultedin requests from grain buyers that grain be tested for thisorganism. In a recent survey of bacteria on milling wheat fromCanada Salmonella spp. and E. coli were not detected, butcoliform bacteria have been found in about 25% of samples ofwestern milling wheat (Blaine Timlick, Canadian Grain Com-mission, personal communication).

Most studies of pathogenic bacteria in grain have used con-ventional, culture-based methods (Eyles et al., 1989; Richteret al., 1993; Berghofer et al., 2003). Those methods are time-consuming and have low accuracy. Polymerase chain reaction(PCR) technology has proven to be valuable for the detection ofbacteria in foods. With its high levels of sensitivity and speci-ficity, PCR can be used for the rapid detection of pathogenicbacteria contaminating various foods. Multiplex PCR assaysemploy multiple sets of primers to amplify more than one targetsequence simultaneously in a single reaction. Multiplex PCRassays have been used to detect and/or identify one organism byamplification of more than one gene, or multiple organisms canbe detected simultaneously by targeting unique sequences fromeach organism (Fratamico, 2001).

The aim of this studywas to establish a rapid and simplemethodfor simultaneous detection of E. coli, L. monocytogenes and S.typhimurium in artificially inoculated wheat grain using PCR.

2. Materials and methods

2.1. Bacterial strains and preparation of inoculum

Salmonella typhimurium (# 03-5608), S. agona (# 03-0890),and S. hadar (# 03-4494) were originally from the NationalMicrobiology Laboratory (Canadian Science Centre for Humanand Animal Health, Winnipeg, MB, Canada). A non-pathogenicstrain of E. coli was from the Food Product DevelopmentCenter, Portage la Prairie, MB, Canada, and L. monocytogenes(# 19112) was from the American Type Culture Collection,Manassas, Virginia, USA. All isolates were provided by Dr.Greg Blank of the Department of Food Science, University ofManitoba, Winnipeg, MB, Canada.

Table 1List of primer sequences, expected DNA fragment length and sources of primers

Organism Primer name Sequence (5′–3′)

E. coli GADA/BF ACCTGCGTTGCGTAAATAGADA/BR GGGCGGGAGAAGTTGAT

L. monocytogenes LM404/F ATCATCGACGGCAACCTCLM404/R CACCATTCCCAAGCTAAA

Salmonella spp. SalinvA139 GTGAAATTATCGCCACGTSalinvA141 TCATCGCACCGTCAAAGG

E. coli, L. monocytogenes and S. typhimurium were used asreference/control strains in this study. Cultures of E. coli, L.monocytogenes, S. typhimurium, S. agona and S. hadar werestarted from freezer stocks and grown on either Luria–Bertani(LB) agar medium (1% tryptone, 0.5% Yeast extract, 1% NaCl)or Trypticase Soy Agar medium (Trypticase Soy Broth —Becton Dickinson and Company, MD, USA; plus 1.8% agar).Following overnight incubation at 37 °C, a single colony wasselected and inoculated into 50 mL of LB broth or TrypticaseSoy Broth in a 500 mL Erlenmeyer flask. The cells were grownfor 20 to 22 h at 37 °C with shaking at 200 rpm. For E. coli, L.monocytogenes and S. typhimurium final cell numbers of theinoculum used to inoculate the non-autoclaved wheat weredetermined by making 10-fold serial dilutions in 0.85% NaCl,then spreading 100-μL onto each of four plates of Hektoen–Enteric agar (Oxoid, Nepean, ON, Canada) and Modified Ox-ford agar (Oxoid). E. coliwere enumerated by dispensing 1.0 mLof bacterial suspension onto each of three Petrifilm™ plates(3M Co., St. Paul, MN, USA). LB agar was used for enume-rating the natural microflora of the control wheat sample andfor enumerating the target species recovered from the inocu-lated, autoclaved wheat grain. Incubation for the natural mic-roflora was at 25 °C for 48 h, and incubation for the targetbacteria was at 37 °C for 24 h for E. coli and S. typhimurium, orfor 48 h for L. monocytogenes.

2.2. Inoculation of wheat samples

A sample of #1 Canada Western Red Spring wheat grainfrom the 2004 harvest was used for all tests. Equal numbers ofthe three bacteria were used for inoculation of the grain. Grainwas inoculated either with a single species or with all threespecies simultaneously. Media bottles (500 mL) containing25 g of wheat grain were inoculated with bacteria at numbersranging from 1010 to 103, for inoculation of each speciesalone, or 3×1010 to 3×103 for inoculation of the three speciestogether. To assess the utility of the primers for detection of thetarget bacteria in the absence of a natural flora, the wheat grainwas autoclaved for 20 min at 121 °C then dried overnight at37 °C. To assess the suitability of the method for detecting thetarget bacteria when natural microflora were present, the wheatgrain was not autoclaved. The inoculated wheat grain wasvigorously mixed by shaking for about 30 s to distribute thebacteria. The cap was then loosened and the inoculated grainwas allowed to dry in an incubator for one day at 37 °C.Controls consisted of uninoculated samples treated identicallyto the inoculated ones.

Product size (bp) Reference

670 McDaniels et al. (1996)GGGAGAC 404 Wu et al. (2004)CCAGTGCTCGGGCAA 284 Rahn et al. (1992)AACC

Fig. 1. Amplification products obtained by multiplex PCR. M, Low mass DNAladder (Invitrogen, CA); 1, negative control; 2, 3 and 4 PCR with E. coli; L.monocytogenes and S. typhimurium DNA (100 pg each), respectively. 5, PCRwith 100 pg DNA from each of E. coli and S. typhimurium, and 6, PCR with100 pg DNA from each of E. coli, L. monocytogenes, and S. typhimurium.

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2.3. Enumeration of bacteria from inoculated wheat samples

After drying the inoculated grain, 225 mL of freshly madebuffered peptone water (10 g peptone mixture, 5 g sodiumchloride, 3.5 g di-sodium hydrogen phosphate and 1.5 g potas-sium di-hydrogen phosphate per litre) was added to each bottlecontaining autoclaved or non-autoclaved grain. To suspend thebacteria, the bottles were shaken for 10 min at 200 rpm. Forenumeration of bacteria, 0.5 mL of each rinse fluid was seriallydiluted with 4.5 mL volumes of saline solution. The sameselective media and protocol described in Section 2.1 were usedfor enumeration of bacteria. A 1.5 mL portion of rinse fluid alsowas removed from each bottle and placed in a sterile 2 mL

Table 2Enumeration of E. coli, L. monocytogenes and S. typhimurium on non-autoclaved, inoone day enrichment when all three organisms were inoculated together

Dilutionfactor

Experiment a E. coli

Number b (CFU/mL) PCR c

100 A (6.42±0.45)×106 +B (1.10±0.17)×106 +

10−1 A (7.25±3.00)×105 +B (2.00±1.00)×104 +

10−2 A (2.88±0.34)×104 +B (1.67±0.58)×103 +

10−3 A (7.75±1.02)×103 +B (3.33±2.51)×102 +

10−4 A (2.58±0.33)×103 +B (1.40±0.20)×102 +

10−5 A (1.87±0.13)×103 +B (7.33±1.15)×101 +

10−6 A (3.90±0.52)×102 +B (5.66±2.52)×101 +

10−7 A ND −B ND −

a Experiments A and B were carried out in Dec. 2005 and January 2006, respectib The number was counted after drying the grain overnight in an incubator at 37 °C

of four replications for L. monocytogenes and S. typhimurium and three for E. coli).c PCR analysis was carried out after 24 h culture enrichment. + = Presence of PC

microcentrifuge tube for DNA extraction. The bottles and theinoculated agar plates were then incubated at 37 °C overnight.

2.4. Enrichment procedure

After removal of fluid samples for enumeration of bacteriaand DNA extraction without enrichment, the bottles were incu-bated at 37 °C for 24 h. After 24 h, the bottles were again shakenfor 10 min at 200 rpm and a 1.5 mL portion of rinse fluid waswithdrawn from each bottle for DNA isolation.

2.5. DNA isolation, multiplex PCR conditions and datacollection

Bacterial genomic DNA extraction was performed as de-scribed previously (Rich et al., 2001; Malorny et al., 2003)immediately after collection. Each fluid sample was first heatedat 60 °C for 20 min then centrifuged at 16,000 g for 10 min. Theresulting pellet was washed with 300-μL TE buffer (10 mMTris–HCl, 1 mM EDTA, pH 8.0), resuspended by vortexing,and then centrifuged again at 16,000 g for 10 min. After re-moval of the supernatant the pellet was again washed with300-μL of TE buffer and resuspended by vortexing. The solu-tion was then boiled for 10 min and the lysate was immediatelychilled on ice for 5 min. After centrifugation at 16,000 g for10 min, the supernatant containing DNAwas transferred into asecond tube. DNA was also extracted from pure bacterial cul-tures. This DNA was purified using the Wizard DNA Puri-fication Kit (Cat. #A1120; Promega, Madison, WI, USA). DNAfrom pure bacterial cultures was quantified by fluorometry withPicoGreen reagent (Molecular Probes, Eugene, OR, USA) asrecommended by the manufacturer.

culated wheat grain prior to enrichment, and detection using multiplex PCR after

L. monocytogenes S. typhimurium

Number (CFU/mL) PCRc Number (CFU/mL) PCR c

(2.02±0.11)×107 + (2.42±0.61)×107 +(2.56±0.36)×107 + (9.50±1.61)×106 +(2.10±0.06)×106 + (2.03±0.22)×105 +(2.78±0.30)×106 + (2.35±0.85)×105 +(2.30±0.12)×105 + (4.31±0.20)×105 +(2.53±0.51)×105 + (3.93±1.00)×104 +(2.98±0.28)×104 + (1.04±0.07)×105 +(3.78±0.43)×104 + (1.70±0.11)×104 +(1.83±0.10)×103 + (2.27±0.31)×103 +(1.97±0.05)×103 − (1.90±0.11)×103 +(7.47±1.44)×102 − (2.01±0.30)×103 +(2.65±0.71)×102 − (1.32±0.06)×103 +(1.08±0.19)×102 − (5.40±2.9)×101 +(6.25±3.10)×101 − (7.50±2.38)×102 +(5.00±5.70)×100 − ND +(2.50±1.29)×101 − ND +

vely.and prior to enrichment. Data is expressed as mean±standard deviation (average

R product, and − = Absence of PCR product.

24 J. Kim et al. / International Journal of Food Microbiology 111 (2006) 21–25

The primers chosen for the multiplex PCR were determinedafter first evaluating several previously described primers. Oli-gonucleotide primers used for this study are shown in Table 1.The 25-μL multiplex PCR reaction contained 1× AccuPrime™buffer II (20 mM Tris–HCl, pH 8.4, 50 mM KCl, 1.5 mMMgCl2, 0.2 mM of each of the four dNTPs, thermostableAccuPrime™ protein and 1% glycerol), 0.2 μM salinvA primerset, 0.4 μM GADA and LM404 primer sets and 2.5 units ofAccuPrime Taq DNA polymerase (Invitrogen, Burlington, ON,Canada). An additional 3.0 mM MgCl2 was used for the PCR.For the DNA extracted from inoculated grain samples, 4-μL ofDNA out of 100-μL stock was used directly in the 25-μL PCRreaction. The same PCR condition was used for singleplex PCRanalysis for L. monocytogenes on non-autoclaved wheat. Multi-plex PCR was used for all other analyses, regardless of whetherthe wheat had been inoculated with the three species individuallyor simultaneously. Multiplex and singleplex PCR wereperformed in a 96-well-plate using PTC-200 Thermal Cycler(MJ Research, BioRad Laboratories, Waltham, MA, USA). Thethermal cycling program included an initial 2 min denaturationat 94 °C; and then 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and60 s at 72 °C, followed by a final extension for 7 min at 72 °C.The PCR products (20-μL) were separated by electrophoresis in2% (wt/vol) agarose in 1× TAE containing 0.2 μg/mL ethidiumbromide. Images were recorded with a GelDoc UV gel docu-mentation system (BioRad, Hercules, CA, USA).

3. Results and discussion

All strains produced typical growth when inoculated onto theirrespective selective media. PCR with the salinvA primer set pro-duced a PCR product of the expected size from S. typhimurium, S.agona, and S. hagar (data not shown). As shown in Fig. 1 (lanes 2,3 and 4), a mixture of the three primer pairs in a PCR reactioncontaining a DNA template of a single bacterial pathogen am-plified the expected PCR amplicons.

When multiple target organisms were included in the reac-tion containing the mixture of two or three primer pairs, thecorresponding amplicons of different sizes were observed (Fig.1, lanes 5 and 6). This result showed that each primer pair in themixture was sensitive and specific enough to detect its targetDNA sequence from the DNA mixture of three bacterialspecies. In addition, PCR carried out with 10 pg DNA fromeach of the three bacterial species produced the expected results(data not shown). Non-specific PCR products were not detectedusing the mixture of three primer pairs with the DNA of thethree species.

In general, harvested grain has a low moisture content, and isusually stored under dry conditions. To simulate the normalstorage environment, the inoculated grain was dried beforeattempting to recover the bacteria added to the grain. The non-autoclaved wheat samples are reflective of the conditions thatwould exist in a commercial environment. The numbers ofviable bacteria were lower (N80% death rate) after drying thegrain. This is likely due to the low humidity level causingdesiccation of damaged cells. Although this method resulted inthe death of the majority of bacterial cells that were inoculated

onto the grain, it does more closely mimic the situation of grainin storage.

The detection limit of the three bacteria was based on thenumber of target bacteria after drying. PCR-based methods woulddetect DNA from live as well as dead bacterial cells. Methods todetect viable bacterial cells have been suggested (Mukhopadhyayand Mukhopadhyay, 2002; Rudi et al., 2005). In this study, thePCR-based method was used for detection of organisms afterenrichment of bacterial cultures. DNA from the normal microflorain grain may decrease the sensitivity of multiplex PCR. The de-tection limit after enrichment for E. coli, L. monocytogenes and S.typhimurium inoculated individually onto autoclaved wheat grainwas 3 CFU/mL. In non-autoclaved wheat grain inoculated witheach species individually the detection limits after enrichment were7, 700, and 1 CFU/mL respectively. The detection limit by mul-tiplex PCR after enrichment for E. coli, L. monocytogenes and S.typhimurium inoculated together onto non-autoclaved wheat grainwas 56, 1800 andb54CFU/mL respectively (Table 2). It is possiblethat autoclaving of the wheat grain before inoculation increasedPCR sensitivity because of heat inactivation of PCR inhibitors. Fornon-autoclaved wheat grain inoculated with the three organismstogether, increased sensitivity was achieved for L. monocytogeneswhen singleplex PCR was used instead of multiplex PCR. Forexample, expected DNA fragments were observed for b62 CFU/mLusing only the L.monocytogenes specific primer set (singleplexPCR) instead of multiplex PCR. Fortunately, the multiplex worksbest for the two bacteria that have been reported to occur on grain.The lower sensitivity of themultiplex test forL.monocytogenes canbe addressed by employing the same enrichment process used forthe other two species, but then analyzing the extractedDNAusing asingleplex PCR.

Without enrichment of the bacterial culture, the detectionlimits after inoculation of non-autoclaved wheat with E. coli, L.monocytogenes and S. typhimurium together were 300, 30,000and 17,000, respectively. However, enrichment for 24 h in-creased the detection limit of the PCR for all bacteria tested asdescribed above, and would be the recommended procedure.The background microflora of the non-autoclaved wheat grainenumerated on LB agar was 104 to 106 CFU/mL.

The PCR assay described in this study is a quick and reliablemethod to detect the presence of the three bacterial strains inartificially inoculated grain samples. Use of this sensitive me-thodology will allow for a better assessment of the frequencywith which grain may be contaminated with these importantbacteria than does the traditional spread plate method. Whencombined with enumeration of these organisms in sampleswhere PCR based methods have detected their presence, usefulbaseline information can be compiled to identify issues and toaddress customer inquiries and concerns.

Acknowledgements

We would like to thank Dr. Greg Blank for providing us withthe bacterial strains used in this study; Dr. Sung-Jong Lee forhelpful suggestions for PCR set-up, and Drs. Bill Scowcroft andDaniel Perry for reviewing the manuscript and providing valu-able comments.

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