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Food Microbiology 27 (2010) 645e652

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Food Microbiology

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Prevalence and contamination patterns of Listeria monocytogenesin catfish processing environment and fresh fillets

Bang-Yuan Chen a,1, Rajkumar Pyla b,1, Tae-Jo Kim a, Juan L. Silva a, Yean-Sung Jung b,*

aDepartment of Food Science, Nutrition and Health Promotion, Mississippi State, MS 39762, USAbDepartment of Biochemistry and Molecular Biology, Mississippi State, MS 39762, USA

a r t i c l e i n f o

Article history:Received 9 December 2009Received in revised form19 February 2010Accepted 27 February 2010Available online 9 March 2010

Keywords:Listeria monocytogenesPrevalenceCatfishPulsed-field gel electrophoresisEnterobacterial repetitive intergenicconsensus (ERIC)-PCRContamination sourceGenotyping

* Corresponding author at: Department of BiochemP. O. Box 9650, Mississippi State, MS 39762, USA. Tel.(662)325 8664.

E-mail address: yj29@ra.msstate.edu (Y.-S. Jung).1 Both equally contribute to this work.

0740-0020/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.fm.2010.02.007

a b s t r a c t

Catfish skins, intestines, fresh fillets, processing surfaces at different production stages, chiller water andnon-food contact surfaces were sampled for Listeria monocytogenes and other Listeria species. Among 315samples, prevalence of L. monocytogenes, Listeria innocua and a group of Listeria seeligerieListeriawelshimerieListeria ivanovii was 21.6, 13.0 and 29.5%, respectively. No Listeria grayi was detected in thissurvey. While no L.monocytogenes strains were isolated from catfish skins and intestines, the strains werefoundwith a frequencyof 76.7% in chilled fresh catfishfillets and 43.3% in unchilledfillets. L.monocytogenesand Listeria spp. were also detected in fish contact surfaces such as deheading machine, trimming board,chiller water, conveyor belts at different stages, and fillet weighing table. Among L. monocytogenes, 1/2b(47.0%), 3b (16.0%) and 4c (14%) were the predominant serotypes isolated, whereas 4b, 4e, 1/2c and 1/2awere detected atmuch lower frequencies. Genotype analyses of L. monocytogenes isolates using serotyping,pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-PCRrevealed that chiller water represented an important contamination source of L. monocytogenes in thechilled catfish fillets of two processing facilities, whereas fillet weighing table significantly contributed tothe catfish fillet contamination of the third facility. This study suggests that L. monocytogenes contami-nation in the processed catfishfillets originates from the processing environment, rather than directly fromcatfish. Results from this study can aid the catfish industry to develop a plant-specific proper cleaning andsanitation procedure for equipment and the processing environment designed to specifically targetL. monocytogenes contamination.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Listeria monocytogenes and Listeria spp. are characterized asgram-positive and rod-shaped bacteria. Listeriosis caused byL. monocytogenes is a life-threatening disease in fetuses, newborns,immunocompromised people and the elderly (Schuchat et al., 1991;Hof, 2003). A variety of different food products have been impli-cated in human listeriosis cases and outbreaks (Fleming et al., 1985;McLauchlin et al., 1990; Farber and Peterkin, 1991; Dalton et al.,1997). L. monocytogenes has been reported in catfish and itsfinished raw products (Erdenlig et al., 2000; Chou et al., 2006; ChouandWang, 2006). The contamination of L.monocytogenes onwholecatfish and its processing environment could be the source of

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postprocessing contamination on fresh fillets and ready-to-eatproducts (Hu et al., 2006).

Understanding the contamination routes of L. monocytogenes incatfish and other fish processing industry is crucial for preventingfinal products with L. monocytogenes contamination. The detailedanalysis of L. monocytogenes contamination throughout the entireprocess enables tracing the sources and routes of L. monocytogenescontamination. This involves intensive sampling sites including fish(skin and intestine), fish fillets, outside (the water and surface offish holding tank) and inside the processing plants (processingequipments and surfaces, and non-food contact surfaces) (Chouet al., 2006; Hansen et al., 2006; Hu et al., 2006). Environmentaland equipment sampling at different processing stages has beenfound to be an effective approach of tracing Listeria contaminationin the seafood industry (Hansen et al., 2006; Hu et al., 2006).

Typing of L. monocytogenes isolates is another critical compo-nent of tracking bacterial contamination sources throughout foodsystems (Autio et al., 1999; Johansson et al., 1999; Miettinen et al.,1999; Norton et al., 2001; Hoffman et al., 2003). However, sero-typing of L. monocytogenes is not very discriminatory since almost

Live whole fish

Fish holding tank

Stunning

Deheader

Filleter

Skinner

Trim

Chill

Size grading

Ice and Cooler

Fillet/Fish-holding table

Injection

IQF

Water Glaze

Weighing table

Pack

Freezer

Deheader

Skinner

Trim

Chill

Size grading

Ice and Cooler

Waste

Waste

Whole Dressed Fish

Dressed Fillet

Evisceration station Evisceration station

Fig. 1. Flow diagram for a typical catfish processing operation.

B.-Y. Chen et al. / Food Microbiology 27 (2010) 645e652646

all strains isolated from human, foods and environments belong toa small number of serotypes (Gellin and Broome, 1989; Farber andPeterkin, 1991; Schuchat et al., 1991). Improved discriminationbetween L. monocytogenes isolates has been achieved by moleculartyping methods, such as restriction enzyme analysis (Baloga andHarlander, 1991; Carriere et al., 1991; Wesley and Ashton, 1991),pulsed-field gel electrophoresis (PFGE) (Swaminathan et al., 2001;Gerner-Smidt et al., 2006), multilocus enzyme electrophoresis(Boerlin and Piffaretti, 1991; Harvey and Gilmour, 1994), ribotyping(Baloga and Harlander, 1991), multi-virulence-locus sequencetyping (Zhang et al., 2004), random amplification of polymorphicDNA (Mazurier and Wernars, 1992; Farber and Addison, 1994),repetitive element sequence (REP)-PCR typing and enterobacterialrepetitive intergenic consensus (ERIC)-PCR typing (Gilson et al.,1984; Hulton et al., 1991; Versalovic et al., 1991; Jersek et al.,1999; Chou and Wang, 2006). Of these techniques, PFGE has beenconsidered to be the “gold standard” for subtyping L. mono-cytogenes due to its high reproducibility and discriminatory ability(Swaminathan et al., 2001; Gerner-Smidt et al., 2006). However, itis labor intensive and time consuming. On the other hand, the ERIC-PCR method is relatively simple and cost-effective and shown togenerate DNA fingerprints that allow discriminationwithin a singlebacterial species (Jersek et al., 1999).

In this study, we investigated the prevalence of L.monocytogenesand other Listeria species in catfish and processing environmentand explored the contamination sources of the L. monocytogenesisolates in catfish fillets, based on their genotype determined bymolecular subtyping methods.

2. Materials and methods

2.1. Sampling sites

The samples were collected from catfish (skin, intestine, freshunchilled and chilled fillets), and outside (catfish holding tank andwater) and inside (various processing surfaces, floor and drain) ofthree processing facilities (designated facilities X, Y and Z for thestudy). Flow diagram of a typical processing line and details onsampling site are described in Fig. 1 and Table 1, respectively.

2.2. Sampling, repair, enrichment, and isolationof L. monocytogenes and Listeria spp.

Cells of L. monocytogenes and other Listeria spp. were collected,repaired, enriched and isolated according to the FDA-BAM (Hitchins,1998) with some modifications. Briefly, catfish skin and environ-mental samples were collected by swabbing with 9 cm2 sterileDacron-tipped applicators (Fisher Scientific, Fair Lawn, NJ, USA). Theapplicators were then placed in screw-capped tubes with 1 mlpeptonewater. All samples were transported to the laboratory on icein an insulated container and filled immediately with 9 ml of ListeriaEnrichment Broth (LEB) (Difco, Fisher Scientific). Water samples(240 ml) from fish holding tank were collected in sterilized, screw-capped bottles and transported to the laboratory on ice in an insu-lated container. Fiftymilliliters of thewaterwerefiltered in aNalgene115 ml filter (ApogentTechnologies, Rochester, NY, USA), and 1 mlremaining on the top of the filter was collected and placed ona screw-capped tube to which 9 ml of LEB were added. The catfishfillets were placed in sterile bags and transported to the laboratory.Twenty-five grams of each fillet was aseptically cut, placed in 225mlof LEB and blended for 1 min in a stomacher (Tekmar Company,Cincinnati, OH, USA). Thirtywhole catfisheswere also transported onice in an insulated container. The fishes were aseptically excised and1 g of intestine from each fish was placed in a screw-capped tubecontaining 9 ml of LEB.

All collected samples were incubated at 30 �C for 4 h for therepair and enrichment of Listeria cells. After incubation, the Listeriaselective enrichment supplement (Oxoid, Fisher Scientific) wereadded to each sample and continued to incubate for 20 h at 30 �C.Aliquots of the enriched cultures were streaked onto three OxfordMedium Base agar plates (Difco, Fisher Scientific) containingmodified Oxford Antibiotic Supplement and incubated for 24 h at30 �C. Colonies appearing black were considered to be Listeria spp.Five presumptive Listeria colonies were selected from each plateand further identified by multiplex PCR.

2.3. Multiplex PCR identification of L. monocytogenesand Listera spp.

DNAof isolates thatwere presumptively identified as Listeriawasreleased from cells by lysis using boiling according to Gussow andClackson (1989) with a minor modification. Briefly, 1 ml of over-night culture was centrifuged at 10 000 � g at 2 min and the cellpellet was suspended with 200 ml deionized distilled water. Thesuspension was boiled for 5 min and centrifuged at 10 000 � g for2 min. The resulting supernatant was used as a template DNA formultiplex PCR. Reactionmixture (25 ml) contained 1X GoTaq�GreenMaster Mix (Promega, Madison, WI, USA), each 25 pmol of fourforward primers, 25 pmol of one reverse primer and 2 ml of templateDNA. These four species-specific forward primers (MonoA, Ino2,MugraI, and Siwi2) and one conserved reverse primer (Lis1B) were

Table 1Prevalence of Listeria monocytogenes and other Listeria spp. in catfishes and theirprocessing environment.

Sampling sitea Sourcecode

Number of positive samples(total number of samples tested)

Lmob Linb LseeLweeLivb

Catfish skin A1 0(30) 0(30) 2(30)Catfish intestine A2 0(30) 0(30) 0(30)Fillet before chilling in chiller

water (unchilled catfish fillet)A3 13(30) 12(30) 13(30)

Fillet after chilling in chillerwater (chilled catfish fillet)

A4 23(30) 10(30) 22(30)

Water in fish holding tankoutside processing facility

B1 0(3) 0(3) 0(3)

Surface in fish holding tankoutside processing facility

B2 0(3) 0(3) 0(3)

Deheading machine C1 2(9) 1(9) 0(9)Skinner C2 0(9) 1(9) 0(9)Trimming board C3 3(9) 0(9) 3(9)Chiller water C4 5(9) 3(9) 5(9)Belt after chilling C5 1(9) 1(9) 4(9)Size grading station C6 2(9) 0(9) 1(9)Ice container C7 0(9) 0(9) 1(9)Fish holding tray C8 0(9) 0(9) 5(9)Whole fish holding tray in freezer C9 0(9) 0(9) 1(9)Fillet holding table before injection

and individually quick frozenC10 2(9) 0(9) 7(9)

Conveyor belt after injection andindividually quick frozen (IQF)

C11 1(9) 1(9) 1(9)

Fillet weighing table C12 2(9) 2(9) 4(9)Trimming table for big fish C13 0(9) 0(9) 5(9)Conveyor belt after chilling for

whole dressed fishC14 2(9) 0(9) 0(9)

Container with whole dressed fish C15 0(9) 1(9) 2(9)Whole fish skinner C16 1(9) 1(9) 2(9)Holding table before skinner C17 2(9) 2(9) 2(9)Waste belt C18 2(9) 1(9) 2(9)Freezer wall D1 1(9) 0(9) 0(9)Floor D2 4(9) 3(9) 6(9)Drain D3 2(9) 2(9) 5(9)

Total 68(315) 41(315) 93(315)

a The samples were collected from three processing facilities.b Lmo: Listeria monocytogenes; Lin: Listeria innocua; LseeLweeLiv: Listeria seeli-

gerieListeria welshimerieListeria ivanovii.

B.-Y. Chen et al. / Food Microbiology 27 (2010) 645e652 647

designed by Bubert et al. (1999), based on the conserved andspecies-specific regions of iap gene encoding themajor extracellularproteinp60, and shown to be very effective in differentiating Listeriaspp. into L. monocytogenes, Listeria innocua, Listeria grayi, anda group of Listeria seeligerieListeria welshimerieListeria ivanovii(Bubert et al., 1999; Chen et al., 2009). The amplifications wereperformed in a thermocycler (Eppendorf, New York, NY, USA). Thecycling started with an initial denaturation at 98 �C for 2 min fol-lowed by 30 cycles of a 95 �C denaturation for 30 s, 58 �C annealingfor 30 s and 72 �C elongation for 1 min 30 s. At the end of amplifi-cation, the mixture was subjected to a final extension at 72 �C for5min. The PCRproductswere separated byelectrophoresis on a 1.4%agarose gel. After electrophoresis, the gels were stained for15e20 min in 300 ml ethidium bromide solution (0.5 mg/ml),destained by two washes of 15e20 min with deionized distilledwater, and photographed by BioDoc-itTM Imaging System (UVP,Upland, CA, USA).

2.4. Serotyping

The serotyping of L. monocytogenes isolates was performed bya slide agglutination assay using commercially prepared antisera(Listeria antiserum Seiken kit; Denka Seiken Co., Tokyo, Japan)according to the manufacturer's instruction.

2.5. Pulsed-field gel electrophoresis (PFGE) typing

PFGE typing was performed on the L. monocytogenes isolates(n ¼ 74) using the PulseNet's 30-h protocol for L. monocytogenes(Graves and Swaminathan, 2001) with some modifications. Briefly,each strain of L. monocytogenes was grown overnight in 9 ml oftryptic soy broth (Becton Dickinson, Sparks, MD, USA). Aftercentrifugation, each cell pellet was suspended with TE buffer(10 mM Tris, 1 mM EDTA, pH 8.0) to a density of OD600 ¼ 0.7e0.8.Agarose plugs were made from 1:1 mixture of 1.6% low-meltagarose (Bio-Rad, Hercules, CA, USA) and cell suspensions. Eachplug was lysed in lysis buffer (50 mM Tris [pH 8.0], 50 mM EDTA[pH 8.0], 1% sodium lauryl sarcosine, 0.15 mg/ml protease K) for 2 hat 54 �C, washed 4 times with TE buffer, and digested with 200 U ofApaI (New England Biolabs, Beverly, MA, USA) at 30 �C overnight.The resulting DNA fragments in plugs were separated by electro-phoresis on a 1% agarose (pulsed-field certified agarose, Bio-Rad)gel in 0.5X Tris-borate-EDTA (TBE, 45 mM Tris, 4.5 mM boric acid[pH 8.3], and 1 mM sodium EDTA) buffer in a CHEF-Mapper� XAPFGE (Bio-Rad). The electrophoretic conditions were; initial switchtime, 4 s; final switch time, 40 s; run time, 22 h; pulse angle, 120�;gradient 6 V/cm; temperature,14 �C; ramping factor, linear. Lambdaladder PFGE marker (New England Biolabs) was used as a sizemarker. After electrophoresis, the gels were stained for 15e20 minin 300 ml ethidium bromide solution (0.5 mg/ml), destained by twowashes of 15e20 min with 500 ml deionized water and photo-graphed by BioDoc-itTM Imaging System (UVP). The images weresaved as TIFF files for further analysis.

2.6. Enterobacterial repetitive intergenic consensus(ERIC)-PCR typing

ERIC-PCR typing was performed on the L. monocytogenesisolates (n ¼ 74) using the protocol described by Jersek et al. (1999)with some modifications. Primers ERIC 1R (50-ATG TAA GCT CCTGGG GAT TCA C-30) and ERIC 2 (50-AAG TAAGTGACT GGG GTGAGCG-30) were used (Versalovic et al., 1991). Genomic DNAwas isolatedfrom L. monocytogenes using Wizard� Genomic DNA PurificationKit (Promega) according to the manufacturer's instruction. DNAconcentration was determined at 260 nm using NanoDrop�

ND-1000 UVeVis Spectrophotometer (Thermo Fisher Scientific Inc.,Waltham, MA, USA). PCR mixture (25 ml) contained 1X GoTaq�

Green Hotstart Master Mix (Promega), 25 pmol of each primer and35 ng of template genomic DNA. Amplifications were performedwith a DNA thermocycler (Eppendorf) with temperature rampingas follows: an initial denaturation at 95 �C for 5 min; 33 cyclesat 90 �C for 30 s, at 50 �C for 30 s, at 52 �C for 1 min, at 65 �C for2 min; and a final extension at 65 �C for 8 min. The ERIC-PCRproducts were separated by electrophoresis in a 1.5% agarose gel(14 � 16 mm) in 1X TBE buffer at constant 50 V. After electropho-resis, the gels were stained in 300 ml ethidium bromide solution(0.5 mg/ml), destained in deionized water and photographed byBioDoc-itTM Imaging System (UVP). The images were saved in TIFFfile format for further analysis.

2.7. Clustering analysis of PFGE and ERIC-PCR fingerprints

Analysis of TIFF images was carried out using the BioNumerics�

6.0 software (Applied Maths, Sint-Martens-Latem, Belgium). Simi-larity between fingerprints was determined by the Dice correlationcoefficient at 1% band position tolerance, and dendrograms weregenerated by unweighted pair group method using arithmeticaverage (UPGMA).

B.-Y. Chen et al. / Food Microbiology 27 (2010) 645e652648

3. Results

3.1. Incidence of L. monocytogenes and Listeria spp.in tested samples

Single multiplex PCR differentiated Listeria spp. into L. mono-cytogenes, L. innocua, L. grayi, and a group of L. seeligerieL. welshimerieL. ivanovii, based on the size of amplified product(Bubert et al., 1999; Chen et al., 2009). The L. monocytogenes,L. innocua, L. grayi, and L. seeligerieL. welshimerieL. ivanovii yieldeda PCR product of about 0.66, 0.87, 0.48 and 1.1 kb, respectively (datanot shown). Using this method, prevalence of L. monocytogenes,L. innocua, and L. seeligerieL. welshimerieL. ivanoviiwas determinedto be 21.6 (68 of 315 samples), 13.0 (41 of 315 samples) and 29.5%(93 of 315 samples), respectively, in the 315 samples collected fromcatfish and environmental samples (Table 1). No L. grayi was foundin our survey. While L. monocytogenes and L. innocua strains werenot isolated from catfish skins and intestines, a high incidence ofL. monocytogenes (76.7% [23 of 30 samples]), L. innocua (33.3% [10 of30 samples]), and L. seeligerieL. welshimerieL. ivanovii (73.3% [22of 30 samples]) was detected in chilled fresh catfish fillets.L. monocytogenes (43.3% [13 of 30 samples]) and L. innocua (40% [12of 30 samples]) were also found in fresh fillets before chilling inchiller water. L. monocytogenes and Listeria spp. were found inmanyfish contact surfaces such as holding table, whole fish skinner,trimming board, conveyor belts at different processing stages, andfillet weighing table. L. monocytogenes was also isolated from non-fish contact surfaces such as freezer wall (11.1% [1 of 9 samples])while L. seeligerieL. welshimerieL. ivanovii was found in bulk icecontainer (11.1% [1 of 9 samples]) and fish holding tray (55.6% [5 of9 samples]). L. monocytogenes, L. innocua and L. seeligerieL. welshimerieL. ivanovii were also isolated from floor and drainwith a frequency of 33.3% (6 of 18 samples), 27.8% (5 of 18 samples)and 61.1% (11 of 18 samples), respectively. L. monocytogenes wasfound with a high frequency (55.6% [5 of 9 samples]) in chillerwater, suggesting that chiller water could be an important source ofL. monocytogenes cross-contamination on fillets. L. monocytogenes,L. innocua and the other Listeria spp. were also detected in post-chilling contact surfaces. However, no L. monocytogenes or Listeriaspp. were found outside (surface or water in fish holding tank) theprocessing facility (Table 1).

3.2. Serotyping of L. monocytogenes isolates

Of the L. monocytogenes isolates (n¼ 74), serotypes 1/2b (47.0%),3b (16.0%) and 4c (14%) were frequently isolated, whereas 4b, 4e,1/2c and 1/2a were detected at much lower frequencies (Fig. 2).Unlike a previous report that serotype 1/2awas themost frequently

05015102520353045405

a2/1c2/1e4b4c4b3b2/1

sepytoreS

In

cid

en

ce (%

)

Fig. 2. Incidence of Listeria monocytogenes serotypes in catfish fillets and processingenvironment.

isolated from seafood (Chou and Wang, 2006), serotype 1/2a (1.2%)was barely found in our catfish fillets.

3.3. PFGE typing of L. monocytogenes isolates

PFGE DNA bands within the molecular size range of 10e600 kbof L. monocytogenes isolates (n¼ 74) from each facility were alignedusing clustering algorithm BioNumerics� 6.0, and the resultingmatrices were used to construct dendrograms shown in Fig. 3.Serotype and source code for each L. monocytogenes isolate are alsoshown in the Fig. 3.

Fig. 3A shows the dendrogram of the PFGE fingerprints ofL. monocytogenes strains isolated from facility X. Several isolates(JS29, JS30, JS31, JS33, JS35 and JS36) from chilled catfish filletsproduced identical or almost identical (>95% similarity level) PFGEfingerprints. Their fingerprints were very similar to that of JS27isolate from chiller water. Of these strains, JS31 had a differentserotype (4c), which excludes the possibility of the same clonewiththe others. Isolates JS32 and JS34 from chilled catfish fillets alsoshared identical PFGE fingerprints, and their PFGE patterns werevery similar to those of JS28, another isolate from chiller water andJS37, an isolate from other chilled fillets. Strains JS13 and JS17 wereisolated from conveyor belt after chilling and floor, respectively,and both shared identical PFGE fingerprints. Their fingerprintswere more than 90% similar to that of JS18, an isolate fromunchilled catfish fillets. Another floor isolate (JS16) was also highlyrelated to isolates (JS20 and JS24) from unchilled catfish fillets.Except for JS19 showing a PFGE type identical or almost identicalwith several chilled fillet isolates, none of isolates from unchilledcatfish fillet had similar PFGE type with the ones from chilledcatfish fillets.

In facility Y, some L. monocytogenes isolates (JS50, JS52, JS53,JS54 and JS56) from chilled catfish fillets produced identical ormore than 95% similar PFGE profiles (Fig. 3B). Another isolate (JS47)from chilled catfish fillet yielded a PFGE fingerprint nearly identicalto that of JS40, an isolate from fillet weighing table. Strain JS48,which was isolated from chilled catfish fillet, shared identical PFGEpatterns with JS41, another isolate from fillet weighing table, andwith JS38, an isolate from size grading station. Their fingerprintswere about 90% similar to those of JS51 and JS57, isolates from otherchilled catfish fillets.

Fig. 3C shows the dendrogram of the PFGE fingerprints ofL.monocytogenes isolates from facility Z. Two isolates JS80 and JS81from chiller water shared identical fingerprints. Their fingerprintswere about 95% similar to those of JS82, JS84, JS85 and JS86 isolatesfrom chilled catfish fillets, suggesting that they are geneticallyclosely related. Several isolates from unchilled catfish filletsproduced PFGE types very similar to those of isolates from variousenvironments: JS76 produced a fingerprint identical to that of JS62(grading table isolate), and their fingerprints were also similar tothat of JS60 (trimming board isolate); JS78 yielded a fingerprintalmost similar to that of JS70 (drain isolate). No similar PFGEpatterns were observed in between the isolates from unchilledcatfish fillets and the ones from chilled catfish fillets.

3.4. ERIC-PCR typing of L. monocytogenes isolates

L. monocytogenes isolates (n ¼ 74) were also subtyped by ERIC-PCR method. This method involves the use of primers based onshort repetitive sequence elements that are dispersed throughoutprokaryotes and generates a DNA fingerprint that allows discrimi-nation of bacterial strains (Jersek et al., 1999). DNA bandswithin themolecular size range of 0.5 kbe3 kb were aligned using BioN-umerics� 6.0, and the resulting matrices were used to constructdendrograms shown in Fig. 4.

Fig. 3. Dendrograms of PFGE fingerprints of Listeria monocytogenes isolates from facility X (A), facility Y (B) and facility Z (C). Genetic similarity values between fingerprints werecalculated based on Dice coefficient at 1% band position tolerance, and dendrograms were generated by unweighted pair group method using arithmetic average (UPGMA). For eachfigure, the first column on the right side indicates the number of L. monocytogenes isolates, the second one represents the corresponding serotype, and the last column indicatestheir isolation assigned based on Table 1.

B.-Y. Chen et al. / Food Microbiology 27 (2010) 645e652 649

Fig. 4A shows the dendrogram of ERIC-PCR fingerprints ofL. monocytogenes isolates from facility X. Two isolates (JS21 andJS22) from unchilled catfish fillets shared identical ERIC finger-prints and serotypes. They also gave very similar PFGE fingerprints.These results suggest that JS21 and JS22 may originate from thesame origin of clone. JS37, an isolate from chilled catfish fillet

produced an ERIC-PCR fingerprint more than 90% similar to that ofJS28, an isolate from chiller water. PFGE typing analysis alsoshowed that they were highly related with a cutoff of 90%. Inaddition, JS36, JS29 and JS30, which were isolated from chilledcatfish fillets, yielded ERIC-PCR fingerprints that were more than90% similar to that of JS27, another isolate from chiller water. PFGE

Fig. 4. Dendrograms of ERIC-PCR fingerprints of Listeria monocytogenes isolates from facility X (A), facility Y (B) and facility Z (C). Genetic similarity values between fingerprintswere calculated based on Dice coefficient at 1% band position tolerance, and dendrograms were generated by unweighted pair group method using arithmetic average (UPGMA).

B.-Y. Chen et al. / Food Microbiology 27 (2010) 645e652650

typing analysis also suggested that they were highly related. Nogenotypic similarity between the isolates from unchilled catfishfillets and the ones from chilled catfish fillets, except JS19 and JS23,was observed, which was consistent with the results of PFGE typing(Fig. 3A).

Among the L. monocytogenes isolates from facility Y (Fig. 4B),strains JS48, JS51 and JS57 of chilled catfish fillets was the most

highly related to JS41 that was isolated from weighing table. PFGEtyping analysis also showed that they produced either identical ornearly identical fingerprints. The ERIC pattern of another chilledfillet isolate JS53 was also similar to that of JS40, another isolatefrom weighing table. In facility Y, only one L. monocytogenes (JS46)was isolated from chiller water, but this isolate was shown to be theleast related to L. monocytogenes isolates from chilled catfish fillets.

B.-Y. Chen et al. / Food Microbiology 27 (2010) 645e652 651

PFGE typing analysis also showed poor relatedness between JS46and chilled catfish fillet isolates (Fig. 3B).

Fig. 4C shows the dendrogram of the ERIC-PCR fingerprints ofL. monocytogenes isolates in facility Z. Strains JS62 and JS63, whichwere isolated from grading table and freezer wall, respectively,shared identical ERIC-PCR fingerprints. Two isolates JS82 and JS85from chilled catfish fillets were highly related. Like PFGE typinganalysis (Fig. 3C), the ERIC-PCR typing analysis also revealed thatanother isolate (JS86) from chilled catfish fillets produced a finger-print most similar (>95%) to that of JS81, an isolate from chillerwater. In facility Z, like in facility X, L. monocytogenes isolates fromchilled catfish fillets did not show any genetic similarity to the onesfrom unchilled catfish fillets, which was consistent with the resultsof PFGE typing (Fig. 3C).

4. Discussion

Regardless of processing facility sampled, prevalence data indi-cate that Listeria contamination occurs inside the plant while thereis no incidence of L. monocytogenes in fish skin (A1), intestine (A2),and the outdoor environment (i.e. the water [B1] and the surface[B2] of water holding tank) of facility X. Pre-enrichment of catfishfillets in Listeria Enrichment Broth (LEB) for 4 h increased thepercentage of positive L. monocytogenes up to 60%, as comparedwith a previous report that showed only 20% incidence on chilledcatfish fillets in the summer (Chou et al., 2006). Even if whole fishsamples (skins and intestines)werepre-enriched in LEBwithout theListeria selective enrichment supplement to maximally recoverinjured/stressed cells, no Listeria was found in the samples, sug-gesting that the live catfish is not an important source of Listeriacontamination of catfish fillets. Consistent with our results, Hansenet al. (2006) previously reported very low incidence of L. mono-cytogenes in the outdoor environment and fish farms. Ramos andLyon (2000) also reported the absence of Listeria spp. includingL. monocytogenes in whole catfish but reported their presence incatfish fillets, suggesting that the contamination might be associ-ated with catfish processing such as workers, processing equip-ment, surfaces, utensils, packaging materials and air quality in theplants. In the present study, no single L.monocytogeneswas isolatedfrom the skinner (C2) or from the deheading machine (C1), againsuggesting that catfish skin and intestine are not a source ofL.monocytogenes contamination in catfish fillets. On the other hand,trimming board (C3) on which human contact constantly occursshowed 33% incidence of L. monocytogenes (Table 1). Therefore, thecontamination of L. monocytogenes in the trimming board mightcontribute to high incidence of L. monocytogenes on catfish fillets.The PFGE typing analysis of L. monocytogenes isolates from facilityZ (Fig. 3C) showed that one trimming board isolate (JS60) yieldeda fingerprint highly similar to that of JS76, an isolate from unchilledcatfish fillets (A3). However, the same clustering analysis of theERIC-PCR profiles did not support their genotypic relatedness: thesestrains showed a distant relationship, belonging to different groupsdivided at 54% similarity level. Another trimming board isolate(JS61) also produced a fingerprint highly similar to those of severalcatfish fillet isolates (JS82, JS84, JS85 and JS86) but had a differentserotype (4b) with them (4c), suggesting that they could not be thesame origin. Additional comprehensive studies monitoringcontamination of trimming board and finished products usingmolecular subtyping methods are needed to clarify the importanceof trimming board as a source of L. monocytogenes contamination inprocessed fillets. The chiller water was also contaminated withL. monocytogenes, which might lead to the L. monocytogenescontamination on chilled catfish fillets. As shown in Figs. 3 and 4,some L. monocytogenes isolates from chilled catfish fillets (A4) inprocessing facilities X and Z produced DNA fingerprints closely

similar (more than 90%) to those of isolates from chiller water (C4),suggesting that they might have the same origin within a cutoff of90% similarity level. Lack of genotypic similarities between theisolates from chiller water and the ones fromunchilled catfish fillets(A3) further supports the hypothesis that chiller water is animportant source of L. monocytogenes cross-contamination in chil-led catfish fillets (A4) processed in facilities X and Z. One L. mono-cytogenes strain (JS46)was also isolated from chiller water in facilityY. However, its PFGE patternwas completely different from those ofL. monocytogenes isolates from chilled catfish fillets, suggesting thatchiller water could not be the important source of L. monocytogenescross-contamination in chilled catfish fillets processed in facility Y.Instead, several L. monocytogenes isolates (JS48, JS51, JS57 and JS53)from catfish fillets after chilling had fingerprints similar to those ofsome isolates (JS40 and JS41) from weighing table (C12) (Figs. 3Band 4B), suggesting that thisweighing tablemight be amajor sourceof L. monocytogenes contamination in the chilled catfish fillets offacility Y. Even though the daily hygiene practice such as cleaningand sanitation is performed on the fish contact surfaces whereproduct contamination is associated, some of L. monocytogenes canstill be viable on the surface due to Listeria's adaptation capability inlow temperature, nutrient starvation, and environmentally stressfulconditions (George et al., 1988; Cole et al., 1990), which can causethe L. monocytogenes cross-contamination in processed catfishfillets. Previous studies have suggested that the contamination ofL. monocytogenes on processed fish primarily occurs during theprocessing but no specific contamination sites were established incatfish plants (Boerlin and Piffaretti, 1991; Chou et al., 2006; Huet al., 2006). Autio et al. (1999) also pointed out the major contri-bution of brining processing in the contamination of cold-smokedfish. Thus, the chilling process in fresh catfish fillets could be theprimary contamination source of final product.

In the present study, some isolates (for instance, JS18 and JS78)from unchilled catfish fillets (A3) produced PFGE and ERIC-PCRfingerprints similar to those of floor (D2) or drain (D3) isolates (forinstance, JS17 and JS70), suggesting that JS17 and JS18, and JS70 andJS78 may have the same origin. However, the direction of cross-contamination between the unchilled fillet and flour/drain is notclear at this moment.

In this study, 47.0% (39 of 83 samples) of catfish isolates belongto serotype 1/2b, 19.3% (16 of 83 samples) of isolated L. mono-cytogenes strains are serotype 3b, and 16.9% (14 of 83 samples)belong to 4c. Chou and Wang (2006) reported that serotypes 1/2band 3b were predominant in the L. monocytogenes isolates fromfresh catfish fillets. A difference with our results is that theyobserved the highest occurrence of serotype 3b (30.6%) on onlyfresh catfish fillets, whereas serotype 1/2b (47.0%) was the highestin our samples collected from catfish fillets and processing contactsurfaces. This could be due to difference in the sample sites, time ofthe year and/or changes in their sanitation procedures. Boerlin andPiffaretti (1991) also reported that 1/2a and 1/2b were the majorserotypes found on various fish products.

In conclusion, L. monocytogenes and other Listeria spp wereisolated from catfish fillets and processing facilities. They were notfound in catfish skins or intestines but detected in catfish fillets,suggesting that the Listera contamination in catfish fillets mightoriginate from the processing environments rather than catfishitself. In facilities X and Z, chiller water was an important source ofL. monocytogenes contamination of the chilled catfish fillets.However, in facility Y, the weighing table rather than chiller watersignificantly contributed to the L. monocytogenes contamination inthe processed catfish fillets. The results of this study could assistcatfish processors in developing a plant-specific control strategy tominimize possible L. monocytogenes contamination in final catfishproducts.

B.-Y. Chen et al. / Food Microbiology 27 (2010) 645e652652

Acknowledgements

This paper was approved for publication as Journal Article No.J-11726 of the Mississippi Agricultural and Forestry ExperimentStation (MAFES), Mississippi State University. This work was sup-ported in part by theMAFES Project Numbers MIS-371272 andMIS-401090, byUSDA-ARSGrant No. 58-0790-5-137 and byaMAFES SRI.

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