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Food Control 38 (2014) 1e7

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Food Control

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Prevalence and characterization of Listeria monocytogenes isolatedfrom retail-level ready-to-eat foods in South China

Moutong Chen a,b,c, Qingping Wub,c,*, Jumei Zhang b,c, Ze0an Yan b,c, Jun Wang b,c

a School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR ChinabGuangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology, South China (The Ministry- Province Joint Development), PR ChinacGuangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou510070, PR China

a r t i c l e i n f o

Article history:Received 6 June 2013Received in revised form18 September 2013Accepted 24 September 2013

Keywords:Antimicrobial susceptibilityEnterobacterial repetitive intergenicconsensus polymerase chain reactionListeria monocytogenesMost probable numberRandom amplified polymorphic DNA

* Corresponding author. No. 100, Xianlie Zhong Roa510070, PR China. Tel./fax: þ86 20 87688132.

E-mail address: wuqp203@163.com (Q. Wu).

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.09.061

a b s t r a c t

Listeria monocytogenes is an important food-borne pathogen causing meningitis, meningoencephalitisand abortion. To assess the potential risk to consumer health, the presence of L. monocytogenes wasinvestigated using qualitative and quantitative methods. Ten (6.33%) of 158 retail RTE food samples werepositive for L. monocytogenes and the contamination levels were less than 10 MPN/g,while none of 65dairy products was positive for L. monocytogenes. The 37 strains were grouped into five clusters and twosingletons, five clusters and two singletons, and three clusters and one singleton by enterobacterialrepetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and RAPD fingerprint respectively,at similarity coefficient of 80%. The susceptibility test showed that 83.8% were susceptible to 15 anti-microbials; two were penicillin-resistant, and one was multidrug-resistant to kanamycin, tetracycline,sulfamethoxazole, rifampin, gentamycin, penicillin, and ampicillin. Virulent L. monocytogenes thatpossess partial antimicrobial resistance, and serotypes frequently associated with listeriosis wererecovered from RTE foods. Consumers may, therefore, be exposed to potential risks of L. monocytogenesinfection in South China. This study contributed to the prevalence and contamination levels ofL. monocytogenes in RTE foods in South China for the first time, providing baseline information forChinese regulatory authorities to formulate a regulatory framework for controlling L. monocytogenes toimprove the microbiological safety of RTE foods.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Listeria monocytogenes is an important food-borne pathogenand had been isolated from feces of animals, food, and food-processing plants (Miettinen & Wirtanen, 2006). L. monocytogenescan cause severe listeriosis infections resulting in meningitis,meningoencephalitis, septicemia, abortion, and prenatal infectionin individuals with weakened immune systems, such as fetuses,infants, the elderly, and immunocompromised individuals(Siegman-Igra et al., 2002;Wu, Zhang, Chen, & Yin, 2008). Mortalitydue to listeriosis is up to 20e30% in vulnerable groups (Lukinmaa,Miettinen, Nakari, Korkeala, & Siitonen, 2003).

For surveillance or tracing sources of L. monocytogenes, in recentyears, a number of typing techniques, such as pulsed field gelelectrophoresis (PFGE) (Gerner-Smidt et al., 2006), amplified

d, Yuexiu District, Guangzhou

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fragment length polymorphism (AFLP) (Parisi et al., 2010), randomamplified polymorphic DNA (RAPD) (Lawrence, Harvey, & Gilmour,1993), multi-locus sequence typing (MLST) (Parisi et al., 2010),ribotyping (Pagadala et al., 2012), and enterobacterial repetitiveintergenic consensus-polymerase chain reaction (ERIC-PCR)(Chen, Pyla, Kim, Silva, & Jung, 2010), have been developed todifferentiate L. monocytogenes strains. Of these techniques, PFGEhas been considered to be the “gold standard” for subtypingL. monocytogenes due to its high reproducibility and discriminatoryability (Gerner-Smidt et al., 2006). However, PFGE is labor intensiveand time-consuming. In contrast, ERIC-PCR is a relatively simple,highly reliable, and cost-effective method, and was demonstratedto generate clear DNA fingerprints within a single bacterial species(Jer�sek et al., 1999).

As listeriosis may result from ingestion of contaminated foodproducts, the prevalence, quantitative data, and characterization ofisolates in different types of food is important for risk assessment.Given its ability to tolerate wide pH, low temperature, and saltranges, propagation of L. monocytogenes in ready-to-eat (RTE) foodsmay cause potential risk of listeriosis. Many countries have

M. Chen et al. / Food Control 38 (2014) 1e72

formulated a zero-tolerance policy for L. monocytogenes in certainRTE foods (Nørrung, 2000). Generally, additional handling of RTEfoods at the retail level, such as open-air slicing, weighing, andpackaging, may increase the potential risks of cross contamination(Lin et al., 2006). However, presence of L. monocytogenes in RTEfoods at retail level has received less attention in South China andlittle information on the prevalence and contamination levels ofL. monocytogenes are available. The objective of this study was toinvestigate the occurrence of L. monocytogenes in RTE foods anddairy products at retail level in South China both qualitatively andquantitatively, and to characterize L. monocytogenes isolates bygenotyping and phenotyping for risk analysis of L. monocytogenesfrom RTE food chains.

2. Materials and methods

2.1. Sampling procedure

From July 2011 to August 2012, a total of 223 samples of 158 RTEfoods were collected, including roast duck, roast spare ribs, coldvegetable dish in sauce, brine-soaked duck, roasted goose, saltbaked chicken, braised pork, ham, fried drumsticks and 65 dairyproducts. The sampling sites were distributed through 12 differentcities in South China. All of the samples were kept below 4 �Cduring transportation and testing was initiated within 2 h afterreceipt.

2.2. Qualitative detection

For qualitative detection, an enrichment method was used ac-cording to the National Food Safety Standards of China, with minormodifications (Fig. S1). In brief, samples were submitted to quali-tative analysis for L. monocytogenes by homogenizing 25 g of therespective sample and 225 mL LB1 enrichment broth (Huankai,Guangzhou, China), for 30 s in stomacher bags (Huankai, Guangz-hou, China). Homogenates were incubated at 30.0 �C for 24 h;thereafter, 0.1 mL LB1 enrichment culture was transferred to 10 mLLB2 enrichment broth at 30.0 �C for 24 h. A loopful of the LB2enrichment culture was streaked onto Listeria selective agar plates(CHROM-agar, Paris, France) and incubated at 37 �C for 48 h. Threeor four presumptive colonies, which were typically blue in colorwith a white halo, were selected for Gram staining, catalase test,and oxidase test. Confirmation of L. monocytogenes was performedusing the Microgen ID Listeria identification system (Microgen,Camberley, UK) according to the manufacturer’s instructions. Allisolates designated as L. monocytogenes by Microgen ID wereadditionally identified by duplex PCR targeting the genes hly(coding for listeriolysin O) and inlB (internalin B), as previouslydescribed (Xu, Wu, Zhang, Deng, & Zhou, 2009); the primers usedare shown in Table S2.

2.3. MPN method for quantitative analysis

The most probable number (MPN) method described byGombas, Chen, Clavero, and Scott (2003) was adapted for use in thisstudy (Fig. S1). The MPN was determined on the basis of thenumber of positive tube(s) in each of the three sets and MPN table(USDA, 1998; USFDA, 1998).

2.4. Serotyping by multiplex PCR

The serotypes of 37 L. monocytogenes isolates and five referencestrains (Table S1) were determined using multiplex PCR, as previ-ously described by Doumith, Buchrieser, Glaser, Jacquet, andMartin(2004). The primers used are shown in Table S2. This assay

differentiates isolates into five major serovars, each of which rep-resents more than one serotype, including subtype I.1 (which in-cludes serotypes 1/2a and 3a), subtype I.2 (serotypes 1/2c and 3c),subtype II.1 (serotypes 4b, 4d, and 4e), subtype II.2 (1/2b, 3b, and 7),and subtype III (serotypes 4a and 4c).

2.5. Determination of presence of virulence-related genes

Five PCRs were performed to detect the presence of sevenvirulence-related genes in the 37 L. monocytogenes strains and fivereference strains (Table S1); two multiplex PCRs were designed toidentify the actA, iap, plcA, and inlA genes, individual PCRswere carried out for prfA, plcB, mpl genes, respectively. The primersused for virulence-related genes identification are shown inTable S2 (Chen et al., 2009; Furrer, Candrian, Hoefelein, & Luethy,1991; Notermans, Dufrenne, Leimeister-Wächter, Domann, &Chakraborty, 1991; Suárez, González-Zorn, Vega, Chico-Calero, &Vázquez-Boland, 2001).

2.6. Antimicrobial (AM) susceptibility test

Since no resistance criteria exist for Listeria antibacterial sus-ceptibility testing in Clinical and Laboratory Standards Instituteguidelines (Clinical and Laboratory Standards Institute, 2011) fortested AMs, criteria for staphylococci were applied. A panel of 15antimicrobials at the specific concentration per disc were tested:ampicillin (AMP; 10 mg), cephalothin (CEP; 30 mg), chloramphenicol(CHL; 30 mg), ciprofloxacin (CIP; 5 mg), erythromycin (ERY; 15 mg),gentamicin (GEN; 10 mg), kanamycin (KAN; 30 mg), rifampin (RIF;5 mg), doxycycline (DOX, 30 mg), levofloxacin (LEV; 5 mg), penicillin(PEN, 10 U), tetracycline (TET; 30 mg), vancomycin (VAN; 30 mg),sulfamethoxazole with trimethoprim (SXT; 23.75/1.25 mg), andsulbactam/ampicillin (SAM; 10/10 mg) (Oxoid, Basingstoke, UK).Antimicrobial susceptibility tests were performed as in a previousstudy (Kova�cevi�c, Mesak, & Allen, 2012). Staphylococcus aureusATCC 25923 and Escherichia coli ATCC 25922 were used as qualitycontrol strains for this study. Zones of inhibition were measuredwith a precision caliper to the nearest 0.01 mm. Isolates exhibitingresistance to at least two of the antimicrobial agents tested wereconsidered to be multidrug-resistant strains.

2.7. ERIC-PCR

Genomic DNA was extracted from L. monocytogenes using aBacterial Genomic DNA Purification Kit (Dongsheng Biotech,Guangzhou, China) according to the manufacturer’s instruction.Genomic DNA concentration was determined at 260 nm using aNano Drop�ND-1000UVeVis Spectrophotometer (Thermo FisherScientific, MA, USA). The ERIC primers were from Versalovic,Koeuth, and Lupski (1991). ERIC-PCR typing was performed onthe L. monocytogenes strains using the protocol described by Jer�seket al. (1999) with some modifications. The PCR mixture (25-mL)contained 1 unit GoTaq� Hotstart polymerase (Promega, WI, USA),0.6 mM of each primer, 2.5 mM MgCl2, 0.2 mM each dNTP, and40 ng of template genomic DNA. Amplifications were performedwith a DNA thermocycler (Applied Biosystems, CA, USA) with thefollowing temperature profile: an initial denaturation at 94 �C for3 min; 35 cycles each consisting of 30 s at 94 �C, 30 s at 46 �C, 30 sat 49 �C, and 3 min at 72 �C; and a final extension at 72 �C for10 min. The ERIC-PCR products were separated by electrophoresisin a 1.5% agarose gel with Goldview staining (0.005%, v/v), andphotographed using a UV Imaging System (GE Healthcare, WI,USA). The images were captured in TIFF file format for furtheranalysis.

Table 2Results of qualitative and quantitative methods for RTE products.

No. of samples Qualitative method Quantitativemethod (MPN/g)

41 þ �191 þ �206 � 0.36208 þ �359 þ 0.3409 � 0.61427 � 0.3428 � 0.36509 � 0.3678 þ 3.6

M. Chen et al. / Food Control 38 (2014) 1e7 3

2.8. Random amplified polymorphic DNA typing

The 10-mer primers OPM-01 (50-GTTGGTGGCT-30) and UBC155(50-CTGGCGGCTG-30) were designed and tested in other studies(Farber & Addison, 1994; Lawrence et al., 1993). Reaction mixtures(25-mL) were prepared, each containing one unit of Taq polymerase,2.5 mM MgCl2, 0.2 mM each dNTP, 1.6 mM primer, and 40 mgL. monocytogenes DNA template. For optimal amplifications withprimer OPM-01 and UBC-155, PCR was carried out with GoTaq�

HotStart polymerase (Promega, WI, USA) and Taq DNA polymerase(Thermo Fisher, MA, USA), respectively. The reaction mixtures werecycled with a hot-lid cycler (Applied Biosystems, CA, USA) throughthe following temperature profile: an initial 5 cycles at 94 �C for5 min, 35 �C for 5 min, 72 �C for 5 min; then 30 cycles each con-sisting of 1 min at 94 �C, 2 min at 35 �C, and 2 min at 72 �C; and afinal extension at 72 �C for 10 min. Ten microliters of all ampliconswere resolved by electrophoresis in a 2.0% agarose gel with Gold-view staining (0.005%, v/v), and gels were photographed under UVtransillumination (GE Healthcare, WI, USA); images were saved asTIFF format files for genotype analysis.

2.9. Fingerprint data analysis

The observed bands in the gels were evaluated based on thepresence (coded 1) or absence (coded 0) of polymorphic fragmentsfor the ERIC and RAPD products. Cluster analysis was performedwith NTSYS-pc (Version 2.10), a numerical taxonomy and multi-variate analysis software package (Rohlf, 2000), based on Dice’ssimilarity coefficient (SD), with a 1% position tolerance and theunweighted pair group method using arithmetic averages(UPGMA).

3. Results

3.1. Qualitative and quantitative analysis

Ten (6.33%) samples positive for L. monocytogeneswere detectedamong 158 RTE foods, but no L. monocytogeneswas isolated from 65dairy products in South China after qualitative and quantitativeanalyses. As shown in Table 1, among the positive samples, five(6.25%) were from deli poultry samples, four (26.7%) were from coldvegetable dish in sauce samples, and one from roast spare ribs. NoL. monocytogenes strain was isolated from cooked beef or coldnoodles in sauce (Table 1). The MPN values of all seven positivesamples did not exceed 10 MPN/g; that of six of seven positivesamples were less than 1 MPN/g (Table 2). All 37 isolates (Fig. S6)were positive for hly, the gene encoding listeriolysin O, and inlB,which indicated that all of the isolated strains belonged toL. monocytogenes.

3.2. Serotyping by multiplex PCR

Thirty-seven L. monocytogenes isolates from RTE foods and fivereference strains were serotyped by multiplex PCR (Table S1). As

Table 1The occurrence of L. monocytogenes in RTE foods.

Product category Total tests L. monocytogenes No. (%)

Cooked pork 36 1/36 (2.8)Cooked beef 3 0 (0.0)Deli poultry 80 5/80 (6.3)Cold vegetable dish in sauce 15 4/15 (26.7)Cold noodles in sauce 24 0 (0.0)Dairy products 65 0 (0.0)

shown in Fig. S2, except for subtype III, all serotypes were identifiedamong the 37 L. monocytogenes isolates; 19 (51.3%) and 10 (27.0%)L. monocytogenes strains belonged to serovar II.1, and serovar II.2,respectively; serovar I.1 and I.2 had four isolates each. Serovar II.1was the predominant serotype among the RTE isolates.

3.3. Detection of virulence-related genes in L. monocytogenesisolates

All 37 isolates presented in this study were identified asL. monocytogenes, based on biochemical tests and duplex PCR, andwere examined for the presence of seven virulence marker genesby PCR. All 37 strains harbored inlA, plcA, actA, mpl, iap, and prfAsequences (Figs. S7eS11). Except for strain 427-1, all isolates alsoharbored plcB.

3.4. Antimicrobial susceptibility test

The susceptibility tests suggested that 31 of 37 (83.8%) isolateswere sensitive to all antimicrobials tested in the panel, three (8.1%)L. monocytogenes strains were resistance to PEN and two isolateshad intermediate resistance to PEN. Only one isolate exhibited in-termediate resistance to DOX, and three L. monocytogenes strainsshowed intermediate resistance to AMP (Table 3). All of theL. monocytogenes isolates were susceptible to CHL, VAN, LEV, CIP,ERY, and SAM (Table 3). However, it was surprising to note that thestrain 191-1, isolated from a fried drumstick sample, was amultidrug-resistant strain, which had resistance to seven AMs,namely KAN, TET, SXT, RIF, GEN, PEN, and AMP.

3.5. ERIC-PCR

ERIC-PCR resulted in three to eight amplification bands, with asize range from 300 to 2135 bp, which were specific for the 37L. monocytogenes isolates and five reference strains ofL. monocytogenes (Fig. S3). Bands with molecular sizes of 500, 1000,and 1800 bp were common to most isolates. Most of the isolatesfrom the same positive sample yielded similar fingerprint profiles.At a relative similarity coefficient of 65%, the 37 L. monocytogenesisolates and five reference strains fell into three major clusters,designated as A, B, and C according to the different serovars. ClusterA included serovar I.1 (1/2a, 3a) and I.2 (1/2c, 3c); cluster B includedserovar II.1 (4b, 4d, 4e) and II.2 (1/2b, 3b, 7), while cluster C onlyincluded strain 191-1, which had been isolated from a frieddrumstick sample and which had multidrug-resistance to variousantimicrobials. Based on a similarity coefficient of 80%, we identi-fied five clusters and two singletons; only one cluster includeddifferent serovar strains, the other clusters showed genetic simi-larity ranging from 94% to 100% (Fig. 1).

Table 3Results of antimicrobial resistance of L. monocytogenes isolates in the study.

Antimicrobial agents Breakpoints (mm) No. of isolates (%)

Susceptible (S) Intermediate (I) Resistant (R) Susceptible (S) Intermediate (I) Resistant (R)

Kanamycin �18 14e17 �13 36 (97.3) 0 (0) 1 (2.7)Tetracycline �19 15e18 �14 36 (97.3) 0 (0) 1 (2.7)Sulfamethoxazole with trimethoprim �16 11e15 �10 36 (97.3) 0 (0) 1 (2.7)Doxycycline �16 13e15 �12 36 (97.3) 1 (2.7) 0 (0)Chloramphenicol �18 13e17 �12 37 (100) 0 (0) 0 (0)Penicillin �29 e �28 32 (86.8) 2 (5.4) 3 (8.1)Ampicillin �29 e �28 33 (89.2) 3 (8.1) 1 (2.7)Vancomycina �17 15e16 �14 37 (100) 0 0Levofloxacin �19 16e18 �15 37 (100) 0 0Gentamicin �15 13e14 �12 36 (97.3) 0 1 (2.7)Ciprofloxacin �21 16e20 �15 37 (100) 0 0Erythromycin �23 14e22 �13 37 (100) 0 0Cephalothin �18 15e17 �14 36 (97.3) 0 1 (2.7)Rifampin �20 17e19 �16 36 (97.3) 0 1 (2.7)Sulbactam/ampicillin �15 12e14 �11 37 (100) 0 0

a Breakpoint for Entercococcus spp.

M. Chen et al. / Food Control 38 (2014) 1e74

3.6. RAPD genotyping analysis

All of the L. monocytogenes isolates were typed with OPM-01and UBC-155 (Figs. S4, S5); both primers had comparable differ-entiating power in ERIC-PCR (Fig. 1). Using the primer OPM-01, the37 L. monocytogenes isolates and five reference strains weregrouped into five clusters and two singletons (ievii), prevalentclusters were iv, v, and i, comprising 18, 7, and 7 isolates, respec-tively, whereas clusters ii and vi included only one isolate each(Fig. 2A).

Fig. 1. The characterization of L. monocytogenes isolates from RTE foods in South China. NDoxazole with trimethoprim; RIF: rifampin; GEN: gentamycin; PEN: penicillin; AMP: ampici

Three clusters and one singleton were discriminated amongthe 37 L. monocytogenes isolates and five reference strains usingthe UBC-155 primer, at a relative similarity coefficient of 80%;these were designated as clusters I, II, III, and IV. Surprisingly,the 37 L. monocytogenes isolates and five reference strains weregrouped into three clusters at a relative genetic similarity of 59%(Fig. 2B). It was interesting to observe that three clusters weredifferentiated according to serovar, similar to the clusteringobserved with ERIC-PCR genotyping. Cluster a comprised 11strains, all of which belonged to serovar I.1 and I.2; cluster b

: not detect; SUS: susceptibility; KAN: kanamycin; TET: tetracycline; SXT: sulfameth-llin.

Fig. 2. Dendrograms of L. monocytogenes strains from ready-to-eat foods samples based on primer OPM-01(A) and UBC-155 (B).

M. Chen et al. / Food Control 38 (2014) 1e7 5

included 30 strains, which comprised 20 serovar II.1 strainsand 10 serovar II.2 strains, while only one strain belonged tocluster c.

4. Discussion

4.1. Risk analysis

To date, limited information has been available on the preva-lence and contamination levels of L. monocytogenes present in RTEfood in China. To our knowledge, no study on the prevalence andrisk analysis of L. monocytogenes in retail-level RTE foods fromSouth China has been reported yet. Our results showed that 10samples from 158 RTE foods were positive for the presence ofL. monocytogenes, indicating a prevalence of 6.33%. These data wereconsistent with previous studies (Chao, Zhou, Jiao, Qian, & Xu,2007; Okada, Monden, Igimi, & Yamamoto, 2012). Since, at retaillevel in China, RTE foods are normally processed at 100 �C, the MPNvalue of all seven positive samples was less than 10 MPN/g indi-cating that the contamination of L. monocytogenes in those foodproducts may arise from the additional handling and processing,such as open-air slicing, weighing, and packaging. NoL. monocytogenes was isolated from 65 dairy products, which wasconsistent with some studies previously conducted in China (Chaoet al., 2007; Yan et al., 2010), but was different to results from someother countries (Harakeh et al., 2009; Hong et al., 2007). Thissuggested that the occurrence of L. monocytogenes in the dairyproducts was associated with regional differences. Highly virulentL. monocytogenes strains may cause disease even when present atrelatively low concentration (Graves et al., 2005). L. monocytogenes

with several putative virulence genes associated with theinfection cycle may be potentially pathogenic for consumers,especially genes responsible for expression of phosphatidylinositol-specific phospholipase C (PI-PLC), internalin-like proteins, and lis-teriolysin O (Kaur, Malik, Vaidya, & Barbuddhe, 2007; Vázquez-Boland et al., 2001). The results from our study showed that97.3% of the 37 L. monocytogenes isolates harbored all the virulence-associated genes examined, including inlA, prfA, plcA, plcB,mpl, iap,and actA; only one strain lacked plcB. The observation indicatedmost of the isolates may have full pathogenic potential. However,the presence of seven virulence-related genes in a L. monocytogenesisolate does not always imply that it is capable of causing disease(Jiang, Xu, Chen, Shuai, & Fang, 2006; Van Stelten, Simpson,Ward, &Nightingale, 2010). Further studies will, therefore, be required toexamine whether the strains isolated in this study are pathogenic.

4.2. Serotyping

L. monocytogenes are divided into 13 serotypes based on somatic(O) and flagellar (H) antigens, including three lineages, viz. I, II, andIII. Doumith et al. (2004) have developed a rapid multiplex PCR forserotyping of L. monocytogenes, by which the three lineages weredefined into five distinct phylogenetic groups, each correlated withserovars: I.1 (1/2ae3a), I.2 (1/2ce3c), II.1 (4be4de4e), II.2 (1/2be3be7), and III (4ae4c). Since serotypes 3a, 3b, 4d, and 4e wererelatively rare in foodborne L. monocytogenes, serovar I.1, II.1, andII.2 were considered as serotype 1/2a, 4b, and 1/2b, respectively.L. monocytogenes strains have different abilities to cause humanlisteriosis according to their serotypes. Most strains isolated frompatients with outbreak listeriosis to date belong to serotype 4b

M. Chen et al. / Food Control 38 (2014) 1e76

(Gianfranceschi, Gattuso, D’Ottavio, Fokas, & Aureli, 2007; Orsi, denBakker, & Wiedermann, 2011). In the present study, serovar II.1(4be4de4e) was the predominant serotype among strains isolatedfrom RTE foods, which is in accordance with the previous studies(Aurora, Prakash, & Prakash, 2009; Zhang et al., 2007). This resultsuggested that consumers are exposed to potential risks ofL. monocytogenes infection in South China. Because of the poordiscriminatory power of serotyping for clinical isolates, a number ofmolecular typing methods with high discriminatory power havebeen developed for typing L. monocytogenes strains.

4.3. ERIC-PCR

ERIC-PCR is widely applied for typing of foodborne pathogens,such as L. monocytogenes (Chen et al., 2010) and Cronobactersakazakii (Ye et al., 2009). In this study, a total of 37 test and fivereference L. monocytogenes strains were divided into three clusters,according to different lineages, at a similarity level of 65%. Cluster Aincluded five strains belonging to serovar I.1 and six strainsbelonging to serovar II.2, respectively. Cluster B comprised 10strains belonging to serovar II.2 and 20 strains that belonged toserovar II.1, respectively; strain 191-1 was the only isolate in clusterC (Fig. 1). As shown in Fig. 1, identical ERIC-PCR profiles wereobserved for some isolates with different serotypes, as has alsobeen reported in a previous study (Soni, Singh, Singh, & Dubey,2013). This result suggested that the target sequences of DNAamplified by ERIC primers are not serotype-specific. In addition,L. monocytogenes strains belonging to the same serovar could not bedistinguished efficiently by ERIC-PCR; this observation wasconsistent with a previous study conducted by Jer�sek et al. (1999).Thus, it is necessary to apply another molecular typing method forfurther differentiation of L. monocytogenes, although ERIC-PCR waschosen for analysis of genetic diversity.

4.4. RAPD

RAPD is a very simple and rapid molecular subtyping techniquewith high discriminatory power; it has been used as a powerful toolin the differentiation of foodborne pathogens. In the present study,thirty-seven L. monocytogenes strains and five reference strainswere divided into seven and four clusters by OPM-01 and UBC-155primers, respectively, at a similarity coefficient of 80%. Similar toERIC-PCR fingerprint analysis, RAPD analysis could not discriminateL. monocytogenes isolates belonging to serovar II.1 (Fig. 2), whichmay be due to the homogenous nature of serogroup four strains(Aurora et al., 2009). The discriminatory power of RAPD is com-parable to that of ERIC-PCR. However, it was found that a higherdiscriminatory power could be achieved by using a combination ofERIC-PCR and RAPD fingerprint analysis (Fig. 1). It is noteworthythat some isolates belonging to the same serovar, serovar II.1,possessed an identical patternwhen analyzed by the three differenttyping methods, such as strains 191-2, 427-1, and 359-1, whichwere from distinct districts. This implies a potential commonepidemiological lineage among these three different regions (Figs.1and 2).

4.5. Antimicrobial susceptibility

Since the first antibiotic resistant and multidrug-resistantL. monocytogenes strains were isolated from patients in 1988(Poyart-Salmeron, Carlier, Trieu-Cuot, Courtieu, & Courvalin, 1990),antimicrobial resistant strains were commonly recovered fromfood, natural environment, and clinical cases of listeriosis. Sur-veillance of the emerging antimicrobial resistance profiles ofL. monocytogenes in RTE foods is of utmost importance. The results

of this study suggested a low incidence of antimicrobial resistancein L. monocytogenes strains isolated from RTE foods in South China.Three (8.1%) L. monocytogenes strains were resistant to PEN and twoisolates had intermediate resistance to PEN. This resistance waslower than the studies that showed 40% or 90% penicillin-resistancelevels (Harakeh et al., 2009; Srinivasan et al., 2005); these strainshad been isolated from dairy-based products and from the envi-ronment. AMP in combinationwith GEN, or SXT, is necessary for thetreatment of invasive listeriosis infections (Hof, Nichterlein, &Kretschmar, 1997; Schlech, 2000). In the current study, except formultidrug-resistant strain 191-1, no AMP-resistant strain wasidentified. However, three strains exhibited intermediate resistanceto AMP in the present study. These results suggested the potentialfor AMP-resistant strains to emergence under some conditions.Only one multidrug-resistant strain was recovered in the currentstudy, which was isolated from a fried drumstick sample. The ac-quired resistance to TET and CIP may be related to the excessive useof these antimicrobials in animal feed and as a drug of secondchoice in the treatment of human diseases (Harakeh et al., 2009).Several studies have elucidated that antimicrobial resistance maybe acquired by L. monocytogenes via conjugation (Charpentier,Gerbaud, & Courvalin, 1999; Lyon, Berrang, Fedorka-Cray, Fletcher,& Meinersmann, 2008), self-transferable plasmids (Charpentier &Courvalin, 1999; Poyart-Salmeron et al., 1990), and vertical andhorizontal gene transfer (Charpentier et al., 1999). Surprisingly, thismulti-resistant strain exhibited an unique amplified DNA patternby both ERIC-PCR and RAPD typing (Figs. S3eS5), suggesting thatthis multidrug-resistant strain may have acquired resistance byvertical and horizontal gene transfer or from other microorganismsby conjugative mobilization.

5. Conclusions

In summary, the findings of this study suggested that the highprevalence of L. monocytogenes in RTE foods may reflect poor hy-gienic practices at the final processing of RTE foods at retail level.RTE foods may serve as potential vehicles for transmission ofvirulent L. monocytogenes. On the basis of genotype and phenotypeanalyses, the presence of serotype 4b in RTE foods pose a potentialrisk for causing listeriosis in consumers in South China. Chineseregulatory authorities should consider formulating a regulatoryframework for controlling L. monocytogenes to improve themicrobiological safety of RTE foods.

Acknowledgments

Wewould like to acknowledge the financing support of NationalNatural Science Foundation of China (No. U1031003) and the Sci-ence and Technology Cooperation Projects of China (No.2013DFH30070).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.foodcont.2013.09.061.

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