International Journal of Food Microbiology 147 (2011) 105–111

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International Journal of Food Microbiology

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Occurrence and diversity of free-living protozoa on butterhead lettuce

Mario J.M. Vaerewijck a, Koen Sabbe b, Julie Baré a, Kurt Houf a,⁎a Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgiumb Laboratory of Protistology and Aquatic Ecology, Department of Biology, Ghent University, Belgium

⁎ Corresponding author. Tel.: +32 9 264 74 51; fax:E-mail address: Kurt.Houf@UGent.be (K. Houf).

0168-1605/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2011.03.015

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 August 2010Received in revised form 17 March 2011Accepted 23 March 2011

Keywords:Free-living protozoa (FLP)LettuceLight microscopyDenaturing gradient gel electrophoresis(DGGE)DiversityMost probable number (MPN)

The occurrence and diversity of free-living protozoa (FLP) on butterhead lettuce (Lactuca sativa L.) wasinvestigated using four different sampling techniques (washing, swabbing, homogenization, and excising).FLP were recovered from all leaf samples (n=64), and cultures were FLP-positive after 1 week. Identificationof FLP was performed by light microscopy and sequencing of denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments. Bodo saltans, Spumella (-like) spp. and Cercozoa were the most commonheterotrophic nanoflagellates. Amoebae belongedmainly to the Vannellida and Tubulinida. Colpoda steinii andCyclidium glaucoma were the most common ciliates. The total number of FLP on middle leaves estimated bythe Most Probable Number method ranged from 9.3×102 MPN/g to 2.4×105 MPN/g leaf, with flagellates(92 MPN/g to 2.4×105 MPN/g) being more abundant than amoebae (b3 MPN/g to 9.3×103 MPN/g) andciliates (b3 MPN/g to 9.3×102 MPN/g). Washing or rinsing leaves followed by spin-drying in a householdsalad spinner reduced the protozoan number with maximum one log unit. Our survey shows that FLP onlettuce leaves are a common and diverse but largely unexplored group of microorganisms.

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1. Introduction

To date, knowledge of quality, food safety and microbialcontamination of vegetables mainly concerns spoiling and pathogenicbacteria, yeasts and molds, parasitic protozoa, nematodes and viruses(Barth et al., 2009; Nguyen-the and Carlin, 2000). However, there is apaucity of information about free-living protozoa (FLP) on vegetables.Free-living protozoa (i.e., amoebae, flagellates, and ciliates) are single-celled eukaryotic microorganisms which are ubiquitous in aquatic andterrestrial ecosystems, and are one of the main predators of bacteria.Most of the studies about FLP on vegetables were focused on free-livingamoebae. Representatives of the genera Acanthamoeba, Hartmannella,Naegleria and Vannellawere recovered frommushrooms and vegetablessuch as carrots, cauliflower, lettuce, radishes, onions, spinach andtomatoes (Ciurea-Van Saanen, 1981; Gourabathini et al., 2008;Napolitano, 1982; Napolitano and Colletti-Eggolt, 1984; Rude et al.,1984; Sharmaet al., 2004). Thereare limiteddata on ciliate andflagellatediversity on vegetables. Ciliates (Colpoda sp., holotrichs and hypotrichs)were recovered from mushroom surfaces (Napolitano, 1982) but noidentification to species level was performed. Gourabathini et al. (2008)reported high flagellate counts in drained water from spinach andromaine lettuce anda smaller numberof amoebae andciliates indrainedwater fromspinach. Two isolated ciliateswere identified asGlaucoma sp.

and Colpoda steinii. In a previous study, a high diversity of FLPwas foundon surfaces of vegetable trays in domestic refrigerators and it wassuggested that the protozoan diversity recorded may be related to FLPpresent on vegetables (Vaerewijck et al., 2010).

Most FLP are not associated with foodborne illness. However, invitro studies showed that some FLP such as Acanthamoeba spp. andTetrahymena spp. are able to act as hosts for foodborne pathogens andprotect internalized bacteria against adverse conditions such asdesiccation and exposure to disinfectants (Snelling et al., 2006).Gourabathini et al. (2008) documented that the ciliates Glaucoma sp.and Tetrahymena pyriformis isolated from vegetables internalizedEscherichia coli O157:H7 and Salmonella enterica serovar Thompson,and subsequently expelled small vesicles containing these bacteria. S.enterica serovar Thompson entrapped in Tetrahymena sp. vesicleswere protected against low concentrations of calcium hypochlorite(Brandl et al., 2005). In order to evaluate the role and potential risk ofFLP as reservoir or vector of foodborne pathogens, knowledge on theoccurrence and diversity of FLP in food-related habitats (Baré et al.,2009; Vaerewijck et al., 2008; Vaerewijck et al., 2010) and raw foodproducts, such as fresh vegetables, is necessary.

The main objective of this study was to assess the occurrence andprovide an inventory of FLP diversity on leaves of commerciallyavailable lettuce. Four different sampling methods were applied torecover FLP from the leaves. The diversity was determined by lightmicroscopy (morphospecies) and sequencing (phylotypes) of excisedDGGE-separated 18S rRNA gene fragments. In addition, the effective-ness of washing and spin-drying for removing FLP from lettuce leaveswas tested.

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2. Materials and methods

2.1. Determination of diversity

2.1.1. Sample collectionFour lettuce heads and four packaged ready-to-eat salads (separate,

whole leaves) (all butterhead lettuce, Lactuca sativa L. var. capitata)wereobtained in January–February 2010 from different supermarkets andwere processed at the latest 1 day after purchase. The lettuceheadswerepackaged in sealed plastic bags at the moment of purchase. Packagedlettuce headswere chosen to avoid leaf contactwith other lettuce heads.The outermost leaves of lettuceheadswere aseptically removed becausethey often were dirty, were contaminated with soil or were damaged.Three leaf types were selected for analysis: outer leaves (OL, from thesecond layer), middle leaves (ML, from the fifth layer) and youngestleaves (YL, from the inner rosette). The weight (mean±standarddeviation) of an OL, ML and YL was 14.2±1.3 g, 7.6±1.7 g, and 1.8±0.8 g, respectively.When present, visible soil or dirt was removedwith asterile scalpel and the underlying leaf surfacewas excised and discarded.Leaves fromready-to-eat lettuce (RTE-L, 5.5±1.6 g)were removedwithsterile forceps from plastic bags. In total, four leaf categories (OL, ML, YLand RTE-L) were investigated.

2.1.2. Sample processingFour different sampling methods (washing, swabbing, homogeni-

zation, and excising) were applied to whole leaves from each leafcategory, replicated across lettuce heads and ready-to-eat salad bags.This resulted in a total of 64 leaves examined: four OL, four ML andfour YL per lettuce head (n=4) and four leaves per RTE-L bag (n=4).

2.1.2.1. Washing. Each leaf was transferred to a stomacher bag and50 ml Page's amoeba saline (PAS) (120 mg NaCl, 4 mg MgSO4·7H2O,6 mg CaCl2·6H2O, 142 mg Na2HPO4, and 136 mg KH2PO4 in 1 l ofdeionized water) was added. The stomacher bag was then placed on alaboratory shaker for 5 min at 350 rpm. 30 ml wash solution wastransferred to a Petri dish.

2.1.2.2. Swabbing. The upper and under sides of the leaf surfaces wereswabbed with a PAS-moistened sterile cotton wool. The cotton woolwas transferred to a stomacher bag and PAS was added up to a finalweight of 20 g. After 5 min incubation, the cotton wool wassubsequently gently massaged and squeezed. The liquid (18 ml±0.5 ml) was transferred to a Petri dish and PAS was added to a finalvolume of 30 ml.

2.1.2.3. Homogenization. A nine-fold quantity of PAS was added to aweighed leaf and subsequently homogenized for 1 min in a labstomacher blender. 1 ml of the initial suspension (homogenate) wastransferred to a Petri dish and PAS was added to a final volume of30 ml.

2.1.2.4. Excising. Sections of the base, the middle (containing themidrib) and the top of each leaf were excised with a flame-sterilizedcork-borer (1.5 cm diameter). The three leaf disks were transferred toa Petri dish containing 30 ml PAS.

2.1.3. Cultivation and morphological identification of FLPPetri dishes were incubated in the dark at room temperature

(20 °C±2 °C). Cultures were examined microscopically (invertedmicroscope, Olympus CKX41) on day 0, day 1, day 3, day 5 and day 7.Free-living protozoa were identified using taxonomic sources forprotozoan identification (Foissner et al., 1992; Foissner et al., 1991;Foissner andWenzel, 2004; Lee et al., 2005; Page, 1988; Patterson, 1998;Smirnov et al., 2007). Identificationswere additionally verified using themicro*scope website (http://starcentral.mbl.edu/microscope). Organ-isms were classified according to Adl et al. (2005).

2.1.4. DNA extraction and amplificationDNA extraction was performed on day 0 (t0) and on day 7 (t7) for

all leaves. On t0, 2 ml of each wash solution (see Section 2.1.2.1) wascentrifuged (20 min, 20800×g, 4 °C). The upper part (1.5 ml) wascarefully removed and the pellet was suspended in the remaining500 μl supernatant. DNA extraction was performed with the Char-geSwitch genomic DNA micro tissue kit (Invitrogen, Paisley, UnitedKingdom) using a final eluent of 75 μl. On t7, DNA was prepared bytaking 2 ml from the cultures and subsequently processing asdescribed for the 2 ml wash solution. All DNA extracts were storedat −20 °C until analysis.

Primer sets Euk1A–Euk516r-GC (Díez et al., 2001) and F1427GC–R1616 (van Hannen et al., 1999) amplify a fragment of approx. 560 bpand approx. 210 bp of the 18S rRNA gene, respectively. PCRamplification was performed with a PE Applied Biosystems 9700temperature cycler and PCR reactions were performed as describedpreviously (Vaerewijck et al., 2008).

2.1.5. DGGE analysisDGGE was performed using the DCode universal mutation

detection system (Bio-Rad, Hercules, US). The gel consisted ofpolyacrylamide in 1×Tris-acetate–EDTA (TAE) buffer. PCR productsobtained with primer set Euk1A–Euk516r-GC were separated on a30% to 40% denaturing gradient in a 6% polyacrylamide gel(acrylamide/bisacrylamide ratio, 37.5:1; 100% denaturing acrylamidesolution contained 7 M urea and 40% formamide). A 30% to 55%gradient in 8% polyacrylamide gel was applied to generate profilesfrom PCR-products obtained with primer set F1427GC–R1616. DGGEanalysis, excision and sequencing of prominent bands were per-formed as described previously (Vaerewijck et al., 2008). All BLASTsearches (GenBank) were done in May 2010. Closest relatives wereclassified according to Adl et al. (2005).

2.2. Salad spinner experiment

Ten lettuce heads (butterhead lettuce, packed in plastic) werepurchased in April 2010 in different supermarkets. Between exper-iments, the household salad spinner was disinfected with a sodiumhypochlorite solution (50 mg/l free chlorine), rinsed with sterilewater in order to remove disinfectant residues and wiped with papertowel. Ten middle leaves (=fourth and fifth layer of leaves; 8.3±1.4 g) per lettuce head were longitudinally cut in two halves. For eachleaf, one half was used to determine the FLP number withoutprocessing. The remaining 10 halves were divided in two groupsand used for washing or rinsing followed by spin-drying in ahousehold salad spinner.

2.2.1. WashingA nine-fold quantity of PAS was added to the weighed leaf halves

in stomacher bags and subsequently washed for 5 min at 350 rpm ona lab shaker. They were then removed from the bags, drip-dried(drained PAS was collected, see Section 2.2.4) and spin-dried for 15 s.

2.2.2. RinsingFive leaf halves were transferred to the basket of the salad spinner

and rinsed with 200 ml PAS. The leaves were drip-dried (drained PASwas collected, see Section 2.2.4) and spin-dried for 15 s.

2.2.3. Enumeration of FLPA nine-fold quantity of PAS was added to the weighed leaf halves

(unprocessed leaf halves, washed/spin-dried leaf halves and rinsed/spin-dried leaf halves) and subsequently homogenized for 1 min. FLPwere enumerated by the most probable number (MPN) method inmicrotiter plates (Rønn et al., 1995). Leaf homogenates were seriallydiluted (to 10−6) in TSB/PAS (Tryptic Soy Broth diluted 1:1000 inPAS). 1 ml of each dilution was inoculated in a well in triplicate.

107M.J.M. Vaerewijck et al. / International Journal of Food Microbiology 147 (2011) 105–111

Microtiter plates were incubated at room temperature in the dark andwere microscopically inspected after 1 week. The absence or presenceof FLP (total FLP, amoebae, flagellates, and ciliates) in the wells wasrecorded. The MPN (3 tube test) was calculated according to the USFood and Drug Administration online manual (Blodgett, 2006).

2.2.4. Identification of FLPThe wash solution (obtained from Section 2.2.1), rinsing solution

(obtained from Section 2.2.2), drained PAS and liquid present in thesalad spinner after spin-drying were collected in 15 cm Petri dishes.Cultures were incubated in the dark for 1 week at room temperatureand regularly inspected for FLP. Organisms were microscopicallyidentified as described in Section 2.1.3.

2.3. Bacteriological analysis

Theaerobic plate count (APC)wasdeterminedon lettucehead leaves(OL, ML, YL, untreated and treated leaves from the salad spinnerexperiment) and ready-to-eat lettuce. Serial dilutions of the t0 samplesobtained by homogenization (see Sections 2.1.2.3 and2.2.3)were platedon Plate Count Agar (64 475, Bio-Rad, Marnes-La-Coquette, France)using the Eddy Jet spiral plater (IUL Instruments, Barcelona, Spain).Plates were incubated at 30 °C for 48 h.

2.4. Data analysis

The non-parametric Kruskal–Wallis test was used to determinedifferences in number of taxa between the categories of leaves (OL,

Table 1List of free-living protozoa microscopically identified in cultures obtained from lettuce heaspinner experiment (n=10). Classification according to Adl et al. (2005).

Supergroup First rank Second rank Morphospecies

Amoebozoa Flabellinea Cochliopodium Cochliopodium spDactylopodida Mayorella vesperVannellida Ripella platypodi

Vannella simplexTubulinea Tubulinida Hartmannella ver

Saccamoeba sp.Chromalveolata Alveolata Ciliophora Aspidisca lynceus

Cinetochilum maChilodonella unciColpoda steiniiColpoda sp.Cyclidium glaucoGlaucoma scintillGlaucoma sp.Paramecium putrPlatyophrya sp.Tachysoma pellioTrachelophyllumVorticella convallVorticella sp.

Cryptophyceae Goniomonadales Goniomonas trunStramenopiles Chrysophyceae Spumella cylindri

Spumella-like flaLabyrinthulomycetes Diplophrys sp.

Excavata Euglenozoa Euglenida Notosolenus sp.Peranema trichopPetalomonas sp.

Heterolobosoae Vahlkampfia sp.Kinetoplastea Bodo saltans

Bodo sp.Rhynchomonas n

Opisthokonta Mesomycetozoa Nucleariida Nuclearia sp.Rhizaria Cercozoa Cercomonadida Allantion tachypl

Cercomonas spp.Incertae sedis Eukaryota Centrohelida Heterophryidae Heterophrys sp.

a Three different morphotypes were distinguished.b Five different morphotypes were distinguished. In cultures obtained from ready-to-eat

ML, YL, and RTE-L) and between the different sampling methods.Estimated protozoan numbers (MPN/g) obtained from the salad-spinner experiment were log transformed before statistical analysis. Apaired t-test was performed to compare cell numbers betweenuntreated and washed/spin-dried leaves, and to compare the numberbetween untreated and rinsed/spin-dried leaves. A probability ofPb0.05 was required for statistical significance.

3. Results

3.1. Microscopic analysis of FLP diversity

Table 1 compiles all 41 organisms which were identified to thegenus or species level in all cultures throughout the whole incubationperiod. A higher diversity was found in the salad spinner experimentwhich may be due to the higher number of lettuce heads (n=10)examined in this experiment. Heterotrophic nanoflagellates includedChrysophyceae, amoeboid and bodonid flagellates. The most com-monly encountered flagellates were Bodo saltans and Spumella(-like)species which became abundant in most cultures after 3 days.Amoebae included monopodial (Hartmannella, Saccamoeba), eruptive(Vahlkampfia sp.), mayorellian (Mayorella vespertilioides) and fan-shaped (Vannella simplex, Ripella platypodia) morphotypes. Vannellidaand Tubulinida were the most common amoebae. The ciliates C. steiniiand Cyclidium glaucoma were the most common ciliates. Fouramoebae, twenty-one flagellates and two ciliates could not beidentified to the genus or species level. Besides FLP, the microalgaeChlamydomonas sp. and Desmodesmus sp. were detected.

ds (n=4), ready-to-eat lettuce packages (n=4) and lettuce heads used in the salad

Four sampling methods Salad spinner experiment

Lettuce heads Ready-to-eat lettuce Lettuce heads

. + +tilioides +a + +

+ +miformis +

+ ++

rgaritaceum +nata +

+ + ++

ma + +ans + +

+inium +

+nellum +sp. +aria complex + +

+cata + +ca + + +gellatesa + + +

++

horum ++ +

++ + ++ +

asuta ++

oon + +b + + +

+

salads, two morphotypes were found.

108 M.J.M. Vaerewijck et al. / International Journal of Food Microbiology 147 (2011) 105–111

3.2. Molecular analysis of FLP diversity: DGGE profiling and sequencingof selected DGGE-bands

A total of 160 PCR products obtained from 16 t0 extracts and 64 t7extracts (each amplified with both primer sets) were subjected toDGGE. In 22 PCR products no bands were detected (17 for primer setEuk1A–Euk516r-GC and 5 for primer set F1427GC–R1616). Thenumber of bands per lane obtained with primer sets Euk1A–Euk516r-GC and F1427GC–R1616 ranged from 1 to 12 and 1 to 13,respectively, with usually only 1 or 2 intense bands. From five t0profiles (n=1 for Euk1A–Euk516r-GC; n=4 for F1427GC–R1616),five bands were excised whose sequences affiliated with protozoansequences. In four other t0 profiles (n=3 for Euk1A–Euk516r-GC;n=1 for F1427GC–R1616), faint bands were present which increasedin band intensity in the corresponding t7 profiles; sequences of theset7 bands were also affiliated with protozoan sequences.

From 33 selected bands obtained with primer set F1427GC–R1616, 22 sequences were affiliated with Chrysophyceae (mainlySpumella-like flagellate JBM/S11), six with Cercozoa, one sequencewith Ciliophora and one with Heterophryidae. Three sequences wereaffiliated with fungi. From 35 selected bands obtained by Euk1A–Euk516r-GC, 21 sequences were affiliated with Cercozoa, five withCiliophora, four with Chrysophyceae, one with Heterophryidae andone with Apicomplexa. Three sequences were affiliated with fungi.Five phylotypes (Cercomonas plasmodialis, Chlamydaster sterni,C. glaucoma, Spumella sp. and Spumella-like JBM/S11) were identifiedby both primer sets. Although the sequences with the highest BLASTscores often matched uncultured protozoan sequences, the mostclosely related organisms are presented in Table 2. Remarkably,vannellid amoebae and the flagellate B. saltans were frequentlyobserved in enrichment cultures but were never retrieved usingDGGE.

3.3. Differences related to sampling methodology and leaf category

Microscopy showed that cultures obtained from all sampled leaves(n=64) using four different sampling techniques were FLP-positive

Table 2Protist phylotypes on lettuce head leaves (n=4) and ready-to-eat lettuce (n=4) as determaccording to Adl et al. (2005).

Supergroup First rank Second rank Mos

Chromalveolata Alveolata Apicomplexa ColpCiliophora Colp

CycGlauNyc

Stramenopiles Chrysophyceae OchOikoSpuSpuSpuSpuSpuUro

Rhizaria Cercozoa Cercomonadida BodCerCercCercHetNeoProtSoilSoilSoil

Silicofilosea AllaIncertae sedis Cercozoa Prol

Incertae sedis Eukaryota Centrohelida Heterophryidae Chla

a A range is given if the sequences retrieved from different samples gave the same close

after one week. In 23 cultures, FLP were observed on t0 mainly afterwashing (n=9) and swabbing (n=7), followed by homogenization(n=4) and excising (n=3). After one day incubation, 62.5% of thecultures were FLP-positive and this number increased to 92.2% after3 days. Amoebae, flagellates and ciliates were recovered from alllettuce head leaves but ciliates were less commonly recovered fromYL. Washing leaves consistently recovered the highest averagenumber of taxa in all leaf categories (Fig. 1), but the differences inFLP diversity retrieved with the different methods for all leafcategories was not statistically significant (P-values ofN0.05). This isquite surprising, as washing and swabbing sample the whole leafsurface, while in homogenization and excising only subsamples of theleaves are incubated, and a lower diversity could therefore beexpected. Irrespective of the sampling methodology, YL and RTE-Lhad an average lower diversity than OL andML but this difference wasstatistically also not significant (P-values of N0.05). The lack ofstatistically significant differences between leaf categories andsampling methods is due to the large variation between lettuceheads and RTE-L bags.

3.4. Salad spinner experiment

The estimated total protozoan numbers for untreated lettuceleaves ranged from 9.3×102 MPN/g to 2.4×105 MPN/g leaf, with 80%of the leaves having protozoan numbers of ≥2.3×104 MPN/g.Flagellates were by far the most abundant protozoan group andshowed numbers (92 MPN/g to 2.4×105 MPN/g) in the same order ofmagnitude as the total FLP numbers. Numbers of amoebae rangedfrom b3 MPN/g to 9.3×103 MPN/g. The lowest number was recordedfor ciliates (b3 MPN/g to 9.3×102 MPN/g). Washing leaves followedby spin-drying reduced the number of FLP, amoebae, flagellates andciliates with approx. 1.0 log, and this difference was statisticallysignificant (P-values of 0.0038, 0.0005, 0.0127, and 0.0108, respec-tively) (Fig. 2). Rinsing leaves followed by spin-drying had less effect(max. 0.5 log reduction) and the difference between untreated andtreated leaves was not significant (P-values of 0.06, 0.07, 0.08 and 0.06for total FLP, amoebae, flagellates and ciliates, respectively).

ined by sequencing of DGGE-separated partial 18S rRNA gene sequences. Classification

t closest related organism Accession number Percentage similaritya

odellidae sp. GQ411073 96.0oda sp. AY905498 100lidium glaucoma EU032356 100comides bromelicola AJ810077 99.8totheroides parvus AF145352 64.4–66.0romonas sp. FN429125 100monas sp. AY520451 100mella sp. AB425951 98.5–100mella-like flagellate JBM/S11 EF043285 99.3–100mella-like flagellate JBAS37 AY651094 96.6mella-like flagellate JBNA45 DQ388541 96.1mella-like flagellate JBNZ43 AY651095 100glena americana EF165131 100omorpha minima AF411276 89.1–92.9cozoa sp. EU709138 100omonas paraglobosa FJ790698 100omonas plasmodialis AF411268 99.0–99.3eromita globosa U42447 95.3–95.9cercomonas sp. AY884335 99.6aspis grandis DQ303924 91.8flagellate AND21 AY965866 96.8–100flagellate AND24 AY965867 100flagellate AND25 AY965868 97.3s sp. AF411263 99.4eptomonas faecicola AF411275 91.1mydaster sterni AF534709 89.3–98.0

st relative but a different sequence similarity.

Fig. 1. Average number of protozoan taxa observed in cultures during an incubation period of 7 days. Cultures obtained after applying four sampling techniques (washing (W),swabbing (S), homogenization (H), and excising (E)) on lettuce head leaves and ready-to-eat lettuce. OL: outer leaves, ML: middle leaves, YL: youngest leaves, RTE-L: ready-to-eatlettuce. Error bars represent standard deviations.

109M.J.M. Vaerewijck et al. / International Journal of Food Microbiology 147 (2011) 105–111

3.5. Bacteriological analysis

Leaves of lettuce heads were heavily contaminated and the APCranged from 1.5×106 CFU/g to 4.3×107 CFU/g. Only on one YL, alower APC was recorded (3.7×102 CFU/g). High bacterial numberswere also obtained from ready-to-eat lettuce with APC ranging from6.5×106 CFU/g to 1.7×107 CFU/g.

The APC of untreated, spin-dried washed and spin-dried rinsedleaves ranged from 3.4×106 CFU/g to 7.7×107 CFU/g, 3.0×106 CFU/gto 1.5×107 CFU/g, and 1.6×106 CFU/g to 7.6×107 CFU/g, respectively.

4. Discussion

This study shows that free-living protozoa (FLP) are common onleaves of commercially available lettuce. Motile FLP observed in t0cultures indicated that an active protozoan community was presenton lettuce leaves. Some species were only detected after a few dayssuggesting that they were present as cysts (Corliss, 2001; Corliss andEsser, 1974; Gutiérrez et al., 2001) or in low numbers. The totalnumber of FLP on unprocessed middle leaves reached up to2.4×105 MPN/g leaf. Microscopy and sequencing of partial 18SrRNA gene fragments revealed the dominance of Spumella(-like)

Fig. 2. Effect of washing or rinsing followed by spin-drying on the number of total free-livinglog MPN/g. Error bars represent standard deviations.

flagellates and Cercozoa, small heterotrophic flagellates which areubiquitous in water and soil habitats. The most common phylotypeswere Spumella-like flagellate JBM/S11 and soil flagellate AND21whichare related to Spumella elongata and to Heteromita globosa, respec-tively (Boenigk et al., 2005; Lara et al., 2007). B. saltans was the mostfrequently observed flagellate in cultures. Estimated flagellatenumbers of 2.3×103 MPN/ml to 2.4×105 MPN/ml in wash solutionswere found (data not shown) which is in accordance withGourabathini et al. (2008) who reported high numbers of flagellates(range of 1.1×105 cells/ml to 8.2×106 cells/ml) in water drainedfrom romaine lettuce and spinach. Naked amoebae (mainly Vannel-lida and Tubulinida) were commonly detected using microscopy butwere not found in the sequence data (see below). No Acanthamoebaspp. were detected which is in contrast to the paper of Napolitano andColletti-Eggolt (1984) who found members of this genus as the mostfrequently encountered amoebae. The number of amoebae in thepresent study is in the same range as recorded by Napolitano andColletti-Eggolt (1984) who found several hundreds of amoebae/gramof lettuce leaf. Ciliates were mainly represented by colpodids,hymenostomes and scutociliates. C. steinii and C. glaucoma were themost common ciliates. Both species are ubiquitous ciliates which havealso been found on other plants such as bromeliads (Foissner et al.,

protozoa, flagellates, amoebae and ciliates. Estimated numbers are expressed as mean

110 M.J.M. Vaerewijck et al. / International Journal of Food Microbiology 147 (2011) 105–111

2003), pitcher plants (Rojo-Herguedas and Olmo, 1999), tree barks(Bartošová and Tirjaková, 2008) and vegetables (Gourabathini et al.,2008).

Culture-based and molecular techniques each have strengths andlimitations (Berney et al., 2004; Crosby and Criddle, 2003; Forneyet al., 2004; Hong et al., 2009; Nocker et al., 2007; Smirnov, 2003,2007; Smirnov and Brown, 2004; von Wintzingerode et al., 1997). Onone hand, many FLP resist cultivation in the laboratory, andmorphology-based identification is often difficult, time-consumingor problematic due to the rarity and/or small size of many species(Smirnov, 2007). Other protist taxa (e.g., B. saltans, vannellidamoebae) on the other hand were not retrieved by DGGE but wereabundant in the cultures as observed by microscopy. Possibleexplanations for this discrepancy include sampling errors and/orPCR artifacts. Sampling errors can arise if the organisms (e.g.,vannellid amoebae) are strongly attached to the surface of the culturerecipient and as a result are underrepresented in the subsamplestaken for DNA extraction. In addition, as mainly prominent bands onDGGE gels were excised and sequenced, it is possible that organismsrepresented by weak bands were missed. The dominance of certaineukaryotic species in cultures does not necessarily result in intensebands on DGGE gels, as previously reported for e.g. Euglena mutabilis(Aguilera et al., 2006). Some organisms such as many amoeboidlineages are rarely or never captured by molecular methods forreasons which are yet unknown but may be due to problems withDNA extraction and/or PCR artifacts (Berney et al., 2004; Epstein andLópez-García, 2008). Several DNA extracts failed to be amplified bythe primer set Euk1A–Euk516r-GC. We also observed differencesbetween the two general primer sets used (primer set Euk1A–Euk516r-GC mainly amplified Cercozoa sequences while Chrysophy-ceae were mainly retrieved with F1427GC–R1616), suggesting thatprimer bias does occur. However, as both general eukaryotic primersets used in our study annealed in silico to various B. saltans sequencesavailable in GenBank, it is unlikely that primer bias is the mainproblem for this organism. A recent survey of culture-independentmarine microbial diversity (Not et al., 2009) compared PCR-free(metagenomic dataset) and PCR-based (clone libraries) approachesand found no major biases resulting from PCR steps. Possibly,variation (up to four orders of magnitude) in rRNA gene copy number(Prokopowich et al., 2003; Zhu et al., 2005) might play a role. Taxawith a high rRNA gene copy number can be over-represented in rRNAgene-based surveys. Because of discrepancies between morphology-based and molecular methods, a combination of complementarymethods (Aguilera et al., 2006; Baré et al., 2009; Dopheide et al., 2009;Massana et al., 2004; Savin et al., 2004; Vaerewijck et al., 2008) andprimer sets (Jeon et al., 2008; Stoeck et al., 2006) is recommended inorder to obtain as complete as possible an inventory of FLP diversity.Group-specific primer sets such as the one designed for Kinetoplastida(Rasmussen et al., 2001) might improve detection of bodonids.

Four different sampling methods (washing, swabbing, homogeni-zation and excising) were tested to explore their usefulness for theassessment of the occurrence of FLP on lettuce leaves. All methodswere successful in retrieving FLP from the leaves; all cultures wereFLP-positive after 1 week of incubation. A quantitative comparison ofthe recovery efficiency of the four sampling methods was not possiblefor several reasons. The leaf homogenates were very turbid whichimpeded microscopy of the incubated samples. Therefore, only amuch smaller subsample of the homogenate could be incubated.Excision per definition only samples parts of the leaf. However,despite the fact that only smaller subsamples were used for the lattermethods, no statistically significant differences were observed in thediversity retrieved from the lettuce leaves with the different methods.While there is a tendency for average diversity to be lower with thehomogenization and excision methods, differences between lettuceheads and ready-to-eat lettuce bags were probably too large for thesedifferences to be significant. The latter may also be responsible for the

fact that no differences in species numberwere observed between leafcategories (OL, ML and YL). It is also interesting to point out that whilewashing appears to capture the highest diversity only about 1 log isactually retrieved using this method as the results of the salad spinnerexperiment showed.

The history of cultivation (open field or protected cultivationsystem, soil or soilless cultivation system) of the lettuce heads isunknown. Contamination routes such as air (Kingston and Warhurst,1969; Rivera et al., 1992; Rogerson and Detwiler, 1999; Schlichting,1964), insects (Maguire, 1963) and contact with soil (Foissner, 1999)might contribute to the contamination of lettuce leaves with FLP.Therefore, lettuce heads from the field and obtained from differentseasons and cultivation methods will probably harbor other protozo-an communities. Ready-to-eat lettuce undergoes several processingsteps such as washing and sanitation. Sanitation reduces the naturalbacterial populations of the produce surface with a few log units (Gilet al., 2009). However, bacterial growth after treatment reachingnumbers similar or higher than unwashed leaves has been reported(Allende et al., 2008). It is currently unknown to what extentindustrial washing and sanitation affect FLP populations on producesurfaces. Our results showed that a lab-scale wash or rinse stepfollowed by spin-drying reduced the FLP only to a small extent (≤1log). Also the bacterial count after washing was hardly reduced whichis in accordance with other studies (Houang et al., 1991; Smith et al.,2003).

In conclusion, our study shows that FLP are a common and diversegroup of microorganisms on lettuce leaves. Results of in vitro studiesindicated that FLP on vegetables might play an important role in theecology of foodborne pathogens (Gourabathini et al., 2008). There-fore, further work should focus on the way in which FLP influence thebacterial populations of vegetable surfaces and whether FLP isolatedfrom vegetables are able to act as hosts of foodborne pathogens underfield conditions.

Acknowledgments

This research was funded by a doctoral fellowship of the SpecialResearch Fund (BOF, Bijzonder Onderzoeksfonds, O1J18206) of GhentUniversity. Johan Van Hende is acknowledged for statistical analysis,Andy Vierstraete for sequencing and Pieter Himpens for technicalassistance.

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