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Prevalence and Contamination Patterns of Listeria monocytogenes in Flammulina velutipes Plants Moutong Chen, 1,2, * Qingping Wu, 2, * Jumei Zhang, 2 Weipeng Guo, 2 Shi Wu, 1,2 and Xiaobing Yang 2 Abstract Four mushroom (Flammulina velutipes) production plants were sampled to investigate the prevalence and contamination source of Listeria monocytogenes. Among 295 samples, the prevalence of L. monocytogenes was 18.6%; the contamination appeared to originate from the mycelium-scraping machinery, contaminating both the product and upstream packaging equipment. Of 55 L. monocytogenes isolates, lineages I.1 (1/2a-3a) and II.2 (1/ 2b-3b-7) accounted for 65.5% and 34.5%, respectively. In addition, lineage I.1 formed significantly thicker biofilms than those within lineage II.2, as determined by crystal violet staining and scanning electron microscopy. Genotype analyses of L. monocytogenes isolates using enterobacteria repetitive intergenic con- sensus-polymerase chain reaction, and random amplified polymorphic DNA revealed that the surfaces of mycelium-scraping machinery may serve as the main source of L. monocytogenes contamination in three of the four plants. This study was the first report to explore the potential contamination sources of L. monocytogenes in the mushroom production chain, thereby providing baseline information for adopting prophylactic measures for critical control points during production in mushroom plants to avoid L. monocytogenes contamination. Introduction L isteria monocytogenes is a facultative intracellular foodborne pathogen that infects humans, with a high mortality rate of 20%–30% for vulnerable groups (e.g., elderly, pregnant women, newborns, and immunocompro- mised individuals) (Siegman-Igra et al., 2002; Lyytikainen et al., 2006; Mun ˜oz et al., 2012). This organism can survive and grow under diverse extreme conditions, such as low temperature, high salinity, and a wide pH range (Vazquez- Boland et al., 2001). Thus, food contamination with L. monocytogenes is a public health concern. In recent years, the prevalence of Listeria spp. in mush- rooms has been reported in several countries (Samadpour et al., 2006; Cordano and Jacquet, 2009; Venturini et al., 2011). Although no listeriosis case associated with the con- sumption of fresh mushroom has been reported, several de- veloped countries have formulated standards and policies to minimize the risk of foodborne listeriosis. Some recalls have even occurred because of the contamination of L. mono- cytogenes in mushroom products (Canadian Food Inspection Agency, 2011, 2012; U.S. Food and Drug Administration, 2012). In addition, a listeriosis case caused by salted mush- room heavily contaminated with L. monocytogenes has been reported in Finland ( Junttila and Brander, 1989). We previ- ously found that a ready-to-eat vegetable product contain- ing fresh mushrooms (Fammulina velutipes) contained high numbers of L. monocytogenes (unpublished data). The contamination routes of L. monocytogenes in the mush- room production industry must be elucidated to prevent final products from being contaminated with L. monocytogenes. However, little information is available about the contami- nation sources of L. monocytogenes in mushroom plants. The aim of the present study was to investigate the prev- alence of Listeria spp. and explore the potential contamina- tion sources of L. monocytogenes in mushroom plants. Materials and Methods Sampling and isolation of Listeria spp. Four mushroom plants (hereafter denoted as A, B, C, and D) were investigated in this study. Plants A and C were lo- cated 15 km apart. Plants A(C), B, and D were located ap- proximately 200 km apart from one another. Samples were collected from different phases of production including composting (compost, n = 20); the inoculation room (solid spawn, sterile compost, inoculating machinery surfaces, and inoculation room air, n = 76); the breeding and mycelium stimulation machinery (n = 60); the growing room (atomized water, shelf surfaces, mushrooms, and air, n = 83), and the 1 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China. 2 Guangdong Institute of Microbiology; State Key Laboratory of Applied Microbiology Southern China; Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application; Guangdong Open Laboratory of Applied Microbiology, Guangzhou, China. *Co-first authors: Moutong Chen and Qingping Wu. FOODBORNE PATHOGENS AND DISEASE Volume 11, Number 8, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2013.1727 620

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Prevalence and Contamination Patterns of Listeriamonocytogenes in Flammulina velutipes Plants

Moutong Chen,1,2,* Qingping Wu,2,* Jumei Zhang,2 Weipeng Guo,2 Shi Wu,1,2 and Xiaobing Yang2

Abstract

Four mushroom (Flammulina velutipes) production plants were sampled to investigate the prevalence andcontamination source of Listeria monocytogenes. Among 295 samples, the prevalence of L. monocytogenes was18.6%; the contamination appeared to originate from the mycelium-scraping machinery, contaminating both theproduct and upstream packaging equipment. Of 55 L. monocytogenes isolates, lineages I.1 (1/2a-3a) and II.2 (1/2b-3b-7) accounted for 65.5% and 34.5%, respectively. In addition, lineage I.1 formed significantly thickerbiofilms than those within lineage II.2, as determined by crystal violet staining and scanning electronmicroscopy. Genotype analyses of L. monocytogenes isolates using enterobacteria repetitive intergenic con-sensus-polymerase chain reaction, and random amplified polymorphic DNA revealed that the surfaces ofmycelium-scraping machinery may serve as the main source of L. monocytogenes contamination in three of thefour plants. This study was the first report to explore the potential contamination sources of L. monocytogenes inthe mushroom production chain, thereby providing baseline information for adopting prophylactic measures forcritical control points during production in mushroom plants to avoid L. monocytogenes contamination.

Introduction

L isteria monocytogenes is a facultative intracellularfoodborne pathogen that infects humans, with a high

mortality rate of 20%–30% for vulnerable groups (e.g.,elderly, pregnant women, newborns, and immunocompro-mised individuals) (Siegman-Igra et al., 2002; Lyytikainenet al., 2006; Munoz et al., 2012). This organism can surviveand grow under diverse extreme conditions, such as lowtemperature, high salinity, and a wide pH range (Vazquez-Boland et al., 2001). Thus, food contamination withL. monocytogenes is a public health concern.

In recent years, the prevalence of Listeria spp. in mush-rooms has been reported in several countries (Samadpouret al., 2006; Cordano and Jacquet, 2009; Venturini et al.,2011). Although no listeriosis case associated with the con-sumption of fresh mushroom has been reported, several de-veloped countries have formulated standards and policies tominimize the risk of foodborne listeriosis. Some recalls haveeven occurred because of the contamination of L. mono-cytogenes in mushroom products (Canadian Food InspectionAgency, 2011, 2012; U.S. Food and Drug Administration,2012). In addition, a listeriosis case caused by salted mush-room heavily contaminated with L. monocytogenes has beenreported in Finland ( Junttila and Brander, 1989). We previ-

ously found that a ready-to-eat vegetable product contain-ing fresh mushrooms (Fammulina velutipes) contained highnumbers of L. monocytogenes (unpublished data). Thecontamination routes of L. monocytogenes in the mush-room production industry must be elucidated to prevent finalproducts from being contaminated with L. monocytogenes.However, little information is available about the contami-nation sources of L. monocytogenes in mushroom plants.

The aim of the present study was to investigate the prev-alence of Listeria spp. and explore the potential contamina-tion sources of L. monocytogenes in mushroom plants.

Materials and Methods

Sampling and isolation of Listeria spp.

Four mushroom plants (hereafter denoted as A, B, C, andD) were investigated in this study. Plants A and C were lo-cated 15 km apart. Plants A(C), B, and D were located ap-proximately 200 km apart from one another. Samples werecollected from different phases of production includingcomposting (compost, n = 20); the inoculation room (solidspawn, sterile compost, inoculating machinery surfaces, andinoculation room air, n = 76); the breeding and myceliumstimulation machinery (n = 60); the growing room (atomizedwater, shelf surfaces, mushrooms, and air, n = 83), and the

1School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China.2Guangdong Institute of Microbiology; State Key Laboratory of Applied Microbiology Southern China; Guangdong Provincial Key

Laboratory of Microbial Culture Collection and Application; Guangdong Open Laboratory of Applied Microbiology, Guangzhou, China.*Co-first authors: Moutong Chen and Qingping Wu.

FOODBORNE PATHOGENS AND DISEASEVolume 11, Number 8, 2014ª Mary Ann Liebert, Inc.DOI: 10.1089/fpd.2013.1727

620

harvesting room (package machinery surfaces, scales,conveyor belts, and packaged mushrooms, n = 56). Myce-lium stimulation involves the scraping and smoothing of thefungal mycelium in the neck of a culture bottle priorto fruiting body development and is referred to as themycelium-scraping equipment. For environmental samples,sampling sites were chosen based on both direct contact andproximity to the mycelium or product. This included the airand surfaces of the inoculating machinery, mycelium-scraping machinery, shelves, conveyor belts, scales, andpackaging machinery. Each sampling spot in the productionenvironment was swabbed with nine sterile cotton swabsmoistened with saline solution (0.85%, wt/vol). The cottonswabs were then placed in 225 mL of Listeria enrichmentbroth (Huankai, Guangzhou, China). Air sampling wascarried out using the gravity-plate method. Five Listeria-selective agar plates (CHROM-agar, Paris, France) wereplaced open in each room for 30 min.

All samples were transported to the laboratory on ice inan insulated container and tested within 4 h after receipt.Enrichment and isolation procedures were performed asprevious reported (Chen et al., 2014). For each positivesample, three to five presumptive colonies were confirmed asL. monocytogenes, using the Microgen ID Listeria identifi-cation system (Microgen, Camberley, UK). One of the con-firmed L. monocytogenes isolated from each positive samplewas submitted for further analysis.

Serotyping

The serotypes of 55 L. monocytogenes isolates and fivereference strains (Supplementary Table S1; SupplementaryData are available online at www.liebertpub.com/fpd)were determined using multiplex polymerase chain reac-tion (PCR) as previously described by Doumith et al. (2004a).The primers used are listed in Supplementary Table S2.

Enterobacteria repetitive intergenic consensus (ERIC)-PCR typing and random amplified polymorphic DNA(RAPD) analyses

Genomic DNA was extracted from L. monocytogenesstrains using a Bacterial Genomic DNA Purification Kit(Dongsheng Biotech, Guangzhou, China) according to themanufacturer’s instruction. ERIC primers (ERIC forward: 5¢-ATGTAAGCTCCTGGGGATTCAC-3¢, ERIC reverse: 5¢-AAGTAAGTGACTGGGGTGAGCG-3¢) were designed asdescribed by Versalovic et al. (1991). ERIC-PCR typing wasperformed using the protocol described by Chen et al. (2014).The 10-mer primer UBC-155 (5¢-CTGGCGGCTG-3¢) wasdesigned and tested in a previous study (Farber and Addison,1994). The PCR reaction condition was carried out on the 55L. monocytogenes isolates and 5 reference strains using theprotocol described by Chen et al. (2014).

Cluster analysis was performed with NTSYS-pc (ver-sion 2.10), a numerical taxonomy and multivariate analysis

Table 1. Prevalence of Listeria monocytogenes and Other Listeria spp. in Flammulina velutipes

and F. velutipes Production Environments

No. of positive samples (total samples tested)a

Sampling sites Plant Lmo (%) Listeria spp. (%) Lin (%) Lwe (%) Lgr (%)

Composting phase A 0/5 (0%) 4/5 (80.0%) 4/5 (80.0%) 0/5 (0%) 0/5 (0%)B 0/5 (0%) 3/5 (60.0%) 3/5 (60.0%) 0/5 (0%) 0/5 (0%)C 4/5 (40.0%) 3/5 (60.0%) 3/5 (60.0%) 0/5 (0%) 0/5 (0%)D 0/5 (0%) 4/5 (80.0%) 4/5 (80.0%) 0/5 (0%) 0/5 (0%)

Inoculation room A 0/20 (0%) 0/20 (0%) 0/20 (0%) 0/20 (0%) 0/20 (0%)B 0/20 (0%) 0/20 (0%) 0/20 (0%) 0/20 (0%) 0/20 (0%)C 0/20 (0%) 0/20 (0%) 0/20 (0%) 0/20 (0%) 0/20 (0%)D 0/16 (0%) 0/16 (0%) 0/16 (0%) 0/16 (0%) 0/16 (0%)

Breeding and myceliumstimulation

A 4/15 (26.7%) 6/15 (40.0%) 6/15 (40.0%) 0/15 (0%) 0/15 (0%)B 0/15 (0%) 7/15 (46.7%) 7/15 (46.7%) 0/15 (0%) 0/15 (0%)C 4/15 (26.7%) 5/15 (33.3%) 5/15 (33.3%) 1/15 (6.7%) 0/15 (0%)D 7/15 (46.7%) 5/15 (33.3%) 5/15 (33.3%) 0/15 (0%) 2/15 (13.3%)

Growing room A 3/21 (14.3%) 6/21 (28.6%) 6/21 (28.6%) 0/21 (0%) 0/21 (0%)B 0/24 (0%) 4/24 (16.7%) 4/24 (16.7%) 0/24 (0%) 0/24 (0%)C 0/18 (0%) 7/18 (38.9%) 7/18 (38.9%) 0/18 (0%) 0/18 (0%)D 5/20 (25.0%) 5/20 (25.0%) 5/20 (25.0%) 1/20 (5%) 2/20 (10%)

Harvesting room A 4/12 (33.3%) 5/12 (41.7%) 5/12 (41.7%) 0/12 (0%) 0/12 (0%)B 0/8 (0%) 6/8 (75.0%) 6/8 (75.0%) 0/8 (0%) 0/8 (0%)C 4/10 (40.0%) 7/10 (70.0%) 7/10 (70.0%) 0/10 (0%) 0/10 (0%)D 6/6 (100.0%) 5/6 (83.3%) 5/6 (83.3%) 0/6 (0%) 0/6 (0%)

Products A 5/5 (100.0%) 4/5 (80.0%) 4/5 (80.0%) 0/5 (0%) 0/5 (0%)B 0/5 (0%) 4/5 (80.0%) 4/5 (80.0%) 0/5 (0%) 0/5 (0%)C 4/5 (80.0%) 5/5 (100.0%) 5/5 (100.0%) 0/5 (0%) 0/5 (0%)D 4/5 (80.0%) 5/5 (100.0%) 5/5 (100.0%) 0/5 (0%) 0/5 (0%)

Total 55/295 (18.6%) 100/295 (33.9%) 99/295 (33.6%) 2/295 (0.7%) 4/295 (1.4%)

aLmo, Listeria monocytogenes; Lin, Listeria innocua; Lwe, Listeria welshimeri; Lgr, Listeria grayi.

CONTAMINATION PATTERNS OF L. MONOCYTOGENES 621

software package (Rohlf, 2000), based on Dice’s similaritycoefficient (SD), with 1% position tolerance. The unweightedpair group method was also performed using arithmeticaverages.

Quantitation of biofilm formation

Cells attached onto the well walls were quantified as pre-viously described (Djordjevic et al., 2002), with minor mod-ifications. Six wells per strain were filled with 150 lL of 1%(vol/vol) overnight culture of the bacterium in trypticase soy

broth (TSB), and each microtiter plate included six wells withsterile TSB in control wells. The microtiter plates were tightlysealed and incubated at 25�C for 72 h. After incubation, themedium was removed from each well, and the plates werewashed four times with sterile distilled water to removeloosely attached cells. The plates were then stained with 1%(wt/vol) aqueous crystal violet solution for 45 min and washedfour times to completely remove unbound crystal violet.Thereafter, 150 lL of 95% (vol/vol) ethanol was added to eachwell, and the optical density (OD) at 595 nm was determined.The average OD from the control wells was subtracted from

FIG. 1. The characterization of Listeria monocytogenes isolates from Flammulina velutipes plant environment samples.RAPD, random amplified polymorphic DNA; ERIC-PCR, Enterobacteria repetitive intergenic consensus–polymerase chainreaction; OD, optical density; CMCC, China Medical Culture Collection; ATCC, American Type Culture Collection; SMM,surfaces of mycelium-scraping machinery; PM, packaged mushrooms; PCB, products conveyor belt; SC, scales; COM,compost; MU, mushrooms; SPM, surfaces of package machinery.

622 CHEN ET AL.

the OD of the test wells. The microtiter plate biofilm assayswere performed three times. Comparison of biofilm formationbetween Listeria lineages was analyzed using one-way anal-ysis of variance followed by a Student-Newman-Keuls post-hoc test with the level of significance of p < 0.05.

Biofilm formation assay by scanning electronmicroscopy (SEM)

Six strains of L. monocytogenes strains were selected forthis assay according to the capacity of biofilm formation as

determined by crystal violet, namely, ATCC19115, ABMS-1,AGR-2, AHR-4, DBMS-3, and DGR-1. Glass discs (F 8 mm,WHB Biotech, Shanghai) were placed in a 48-well plate. Eachglass disc was covered with 2 mL of inoculum at 107 colony-forming units/mL in TSB. After 72 h of incubation at 25�C, thegrowth medium was removed and the discs were washed threetimes with 5 mL of sterile distilled water. Thereafter, the discswere fixed with 3% glutaraldehyde for 4 h, dehydrated withgradient ethanol solution, dried with a freeze dryer (HitachiES2030, Japan), and coated with gold–palladium (HitachiE1010, Japan). SEM images were taken using a S-3000NHitachi scanning electron microscope.

Results

Prevalence of L. monocytogenes and Listeria spp.in tested samples

Table 1 shows that among the 295 samples collected fromthe 4 production plants, 55 (18.6%) samples were positive forL. monocytogenes (i.e., the contaminated sites includingcompost, surfaces of mycelium-scraping machinery, mush-rooms, surfaces of package machinery, scales, productsconveyor belts, and packaged mushrooms). No Listeria spp.were found from air samples. Nonpathogenic Listeria spp.(33.9%) exhibited higher frequency in the collected samples.No Listeria seeligeri and Listeria ivanovii were recovered inFlammulina velutipes plants. No sample was found to becontaminated with L. monocytogenes in plant B, while theother three plants were contaminated with L. monocytogenesin different production phases.

Serotyping

Figure 1 shows the lineages of 55 isolates and the sourcesfrom which they were recovered. In samples from plants A,C, and D, 19 (34.5%) of 55 isolates were lineage II.2 (1/2b,3b, and 7), and 65.5% (36/55) were lineage I.1 (1/2a and 3a).

ERIC-PCR typing

The numbers of L. monocytogenes isolates submittedfor genotyping are listed in Supplementary Table S1. ERIC-PCR fingerprints showed 5–10 bands ranging from approxi-mately 190 bp to 2300 bp in size. At the coefficient of 70%,55 isolates fell into three clusters according to the differ-ent lineages. At a relative similarity coefficient of 90%, 55L. monocytogenes isolates were grouped into four clustersdesignated as I, II, III, and IV (Fig. 2).

As shown in Figure 2, cluster III predominated in thesamples collected from plant A, where 47% of the isolateswere recovered from the surfaces of mycelium-scrapingmachinery, mushrooms, and packaged mushrooms. The re-maining nine isolates were divided into two clusters: cluster Iand cluster II. Cluster I included one isolate recovered fromthe surfaces of mycelium-scraping machinery and four iso-lates recovered from harvesting room including scales, con-veyor belts, and surfaces of package machinery. Cluster IIcomprised one isolate recovered from conveyor belts andthree isolates from packaged mushrooms.

In plant C, the 16 isolates were distributed among threeclusters. Eleven isolates from compost, surfaces of myceli-um-scraping machinery, and packaged mushrooms belongedto cluster III. The three isolates recovered from the surfaces

FIG. 2. Dendrogram of Listeria monocytogenes isolatesrecovered from Flammulina velutipes plant samples basedon Enterobacteria repetitive intergenic consensus (ERIC)-polymerase chain reaction. L. monocytogenes isolates werenamed according to the source of isolates; details are shownin Supplementary Table S1. CMCC, China Medical CultureCollection; American Type Culture Collection.

CONTAMINATION PATTERNS OF L. MONOCYTOGENES 623

of mycelium-scraping machinery and two isolates recoveredfrom harvesting room belonged to cluster I and cluster II,respectively.

For the samples from plant D, 22 isolates recovered fromthe surfaces of mycelium-scraping machinery, mushrooms,scales, conveyor belts, and packaged mushrooms exhibitedthe same ERIC-PCR fingerprints and lineage. At a relativecoefficient of 90%, 22 isolates were grouped into cluster IV(Fig. 2).

RAPD typing

A total of 55 isolates collected from plants A, C, and D and5 reference strains were typed with UBC-155 primer. At arelative coefficient of 90%, 55 isolates were grouped into 5clusters and 2 singletons (i–vii). The isolates recovered fromplant A were mainly grouped into clusters i and v. Five iso-lates belonging to cluster i were isolated from the surfaces ofmycelium-scraping machinery, harvesting room, and pack-aged mushrooms (Fig. 1). Cluster v included seven isolatesrecovered from plant A, containing three isolates from thesurfaces of mycelium-scraping machinery, three isolatesfrom mushrooms, and one isolate from packaged mush-rooms. Isolates recovered from the packaged mushroomsbelonged to two lineages and had RAPD patterns identical tothose of isolates from the mycelium-scraping machinery andmushrooms, respectively. In plant C, 16 isolates were dis-tributed among the four clusters. Seven isolates recoveredfrom compost, surfaces of mycelium-scraping machinery,mushrooms, and packaged mushrooms belonged to cluster v.Twenty-two isolates recovered from plant D belonged tocluster vii, which was consistent with the result of ERIC-PCRtyping.

Comparison between typing methods

At the relative similarity coefficient of 70%, the coinci-dence rate between lineage and ERIC-PCR was 100%; wealso observed a good agreement between lineage and RAPD.At the relative similarity coefficient of 90%, when comparing

ERIC-PCR clusters and RAPD clusters, a good agreementwas observed between ERIC-PCR and RAPD (i.e., cluster vand vii typing by RAPD belonged to cluster III and IV ofERIC-PCR, respectively).

Biofilm formation

The abilities of 55 L. monocytogenes isolates and 5 refer-ence strains to form biofilm were tested at 25�C in TSB. Asshown in Figure 1, the quantity of biofilm obtained after 72 hhighly depended on the strain lineage. The OD values oflineage I.1 (serotypes 1/2a and 3a) were significantly higherthan that of lineage II.2 (serotypes 1/2b, 3b, and 7) ( p < 0.01).Surprisingly, several strains belonging to the same cluster asgrouped by ERIC-PCR and RAPD had different abilities toform biofilm. Six strains were selected to further confirm theability of biofilm formation by SEM. Four strains, namely,ATCC 19115, AHR-4, DBMS-3, and DGR-1, formed a three-dimension, organized, dense structure (Figs. 3G and J–L)with individual L. monocytogenes cells surrounded by anexopolymeric substance (Figs. 3A and D–F). Strains ABMS-1 and AGR-2 developed limited biofilms, and a few attachedcells were scattered on the glass surfaces (Figs. 3B, H, C,and I).

Discussion

Recent studies have explored the transmission route ofListeria spp. in the small-scale Agaricus bisporus plant in theUnited States (Viswanath et al., 2013; Weil et al., 2013). Inaddition, our laboratory found a high occurrence of L.monocytogenes in retail-level fresh F. velutipes products inChina (unpublished data). These data may indicate thatconventional disinfection procedures in the production pro-cess did not effectively eliminate Listeria spp., and thisphenomenon may cause cross-contamination in mushroomplants. In the present study, we specifically focused on thecontamination patterns of L. monocytogenes in four mush-room plants and associated environments. Among 295 col-lected samples, 18.6% were positive for L. monocytogenes.

FIG. 3. Micrographs of Listeria monocytogenes biofilms grown in trypticase soy broth at 25�C for 72 h on glass discs byscanning electron microscopy. (A) Strain ATCC19115 (6000 ·); (B) strain ABMS-1 (6000 ·); (C) strain AGR-2 (6000 ·);(D) strain AHR-4 (6000 ·); (E) strain DBMS-3 (6000 ·); and (F) strain DGR-1(6000 ·); (G) ATCC19115 (2000 ·); (H)strain ABMS-1 (2000 ·); (I) strain AGR-2 (2000 ·); ( J) strain AHR-4 (2000 ·); (K) strain DBMS-3 (2000 ·) and (L) strainDGR-1 (2000 ·).

624 CHEN ET AL.

Only plant B was not contaminated with L. monocytogenes.These results indicated that the contamination appeared tooriginate from the mycelium-scraping machinery and wasfound on upstream processing equipment and the packagedmushrooms. These data may indicate that cross-contamina-tion of L. monocytogenes occurred during the productionprocess of mushrooms. About 33.9% of the collected sampleswere positive for nonpathogenic Listeria spp., including L.innocua, L. welshimeri, and L. grayi. No L. ivanovii and L.seeligeri isolates were found in these four plants. L. innocuawas predominant over L. monocytogenes in the plants andassociated samples, which was consistent with a previousfinding (Aguado et al., 2004). Other studies have reportedthat the presence of L. innocua may reduce the detectabilityof L. monocytogenes by both overgrowth and the productionof inhibitory compounds (Cornu et al., 2002; Zitz et al.,2011). Therefore, the presence of L. innocua may be con-sidered as an indicator of contamination with L. mono-cytogenes (Greenwood et al., 2005; Pagadala et al., 2012),indicating that a strict sanitation measure should be adoptedduring the production process.

Serotyping is a useful tool for the epidemiological inves-tigation and tracing of contamination sources. L. mono-cytogenes strains were divided into four lineages (Roberts,et al., 2006; Ward et al., 2008; Orsi et al., 2011), whileDoumith et al. (2004b) grouped L. monocytogenes into threelineages and further defined them into five distinct phyloge-netic groups by rapid multiplex PCR, each correlated with thefollowing lineages: I.1 (1/2a-3a), I.2 (1/2c-3c), II.1 (4b-4d-4e), II.2 (1/2b-3b-7), and III (4a-4c). Given that serotypes 3a,3b, 4d, and 4e are relatively rare in foodborne L. mono-cytogenes, lineages I.1, II.1, and II.2 were considered as se-rotypes 1/2a, 4b, and 1/2b, respectively (Doumith et al.,2004a). Previous study reported that 95% of isolates recov-ered from foods and clinical samples are serotypes 1/2a, 1/2b,1/2c, and 4b (Pontello et al., 2012). In this study, the specificlineage I (1/2a-3a) and II (1/2b-3b-7) of L. monocytogenesdominated in mushrooms plants, which was consistent withthe previous findings on contamination patterns in differentfood-processing environments while only few 4b serotypehas been isolated from facilities (Kathariou, 2002), includingthose for ready-to-eat foods, cull cow and bull processingplants (Gilbreth et al., 2005; Guerini et al., 2007). Con-versely, serotype 4a was found only in a small-scale mush-room-production facility (Viswanath et al., 2013).

L. monocytogenes biofilms can tolerate high concentra-tions of disinfectants and sanitizers, thereby hampering thedecontamination of surfaces (Carpentier and Cerf, 2011; daSilva and De Martinis, 2013). Hence, we were not surprisedthat the strains belonging to lineage I.1 produced signifi-cantly thicker three-dimensional biofilms than those be-longing to lineage II.2. This observation was consistent withprevious reports that L. monocytogenes strains have differ-ent abilities to form biofilms (Borucki et al., 2003; Harveyet al., 2007; Takahashi et al., 2009; Kadam et al., 2013). Inaddition, L. monocytogenes strains belonging to the sameserotype differ in their ability to form biofilms depending onenvironmental factors (Takahashi et al., 2009). Furtherstudies are thus necessary to elucidate the characteristics ofspecific serotypes of L. monocytogenes that dominate pro-duction environments and are resistant to cleaning andsanitation measures.

Packaged fresh mushrooms stored at low temperatures alsoallow the growth and survival of L. monocytogenes (Gon-zalez-Fandos et al., 2001; Folsom et al., 2006). Thus, L.monocytogenes contamination in final products must becontrolled by tracing the pathogen’s sources and routes oftransmission. ERIC-PCR and RAPD are widely used to tracecontamination sources in various food-processing plants(Fonnesbech Vogel et al., 2001; Vogel et al., 2001; Chenet al., 2010; Keeratipibul and Techaruwichit, 2012). In thepresent study, seven isolates recovered from plant A weregrouped into cluster III by ERIC-PCR subtyping at a relativesimilarity coefficient of 90%. These isolates were recoveredfrom the surfaces of mycelium-scraping machinery, mush-rooms, and packaged mushrooms. Samples collected fromthe surfaces of mycelium-scraping machinery were alsopositive for L. monocytogenes in plants C and D (Fig. 1).Surprisingly, only a unique fingerprint was found for theisolates recovered from plant D using both ERIC-PCR andRAPD. These results strongly suggested that the surfaces ofmycelium-scraping machinery may serve as the primarycontamination sources of L. monocytogenes in F. velutipesplants. Among the four plants, L. monocytogenes isolateswere recovered from compost in plant C, suggesting thatraw materials may serve as the origin source of contamina-tion. Similarly, substrate ingredients were the likely pri-mary contamination source in A. bisporus production facility(Viswanath et al., 2013). Long-term comprehensive studiesare needed to trace sources of contamination, and molecularsubtyping methods are a useful measure for identifying per-sistent L. monocytogenes strains at critical control points inmushrooms plants.

Conclusions

Lineage I (1/2a-3a) and II (1/2b-3b-7) were predominant inF. velutipes plants. Genotype and phenotype analyses re-vealed that L. monocytogenes may spread across the entireproduction process of mushrooms plant, from compost to thepackaged mushrooms. The surfaces of mycelium-scrapingmachinery may serve as the primary contamination source ofL. monocytogenes. Strict rules and sanitation measures forcontrolling critical contamination points are necessary toavoid jeopardizing the safety of final products.

Acknowledgments

We thank the staff of the microscopy laboratory at theGuangdong Institute of Microbiology for assisting in ourscanning electron microscopy studies. We gratefully ac-knowledge the financial support of the National Natural ScienceFoundation of China (No. U1031003), the Science and Tech-nology Cooperation Projects of China (No. 2013DFH30070),and Key Projects in the National Science & TechnologyPillar Program during the Twelfth Five-year Plan Period(2013BAD16B05).

Disclosure Statement

No competing financial interests exist.

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CONTAMINATION PATTERNS OF L. MONOCYTOGENES 625

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Address correspondence to:Qingping Wu, PhD

Guangdong Institute of MicrobiologyState Key Laboratory of Applied Microbiology

Southern ChinaGuangdong Provincial Key Laboratory

of Microbial Culture Collection and ApplicationGuangdong Open Laboratory of Applied Microbiology

No. 100, Xianlie Zhong RoadYuexiu District, Guangzhou 510070, China

E-mail: [email protected]

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