Detection of Listeria monocytogenes in Foods and Characterization by PFGE

The aims of the present study were to investigate the prevalence of Listeria monocytogenes in 1042 foods collected from different market to characterize the isolates by phenotypical and molecular methods. In particular, L. monocytogenes obtained from different types of foods such as RTE (kimbap), fish (smoked salmon and seasoned-dried slice fish) and meat (cut raw beef and pork) from 2009 to 2011, were used. Twelve samples (2.1%) were positive for L. monocytogenes. Detection rate of L. monocytogenes varied significantly by food type and ranged from 1.1% to 5.2%. Meat is the highest prevalence for L. monocytogenes (5.2%) followed by RTE (1.8%) and Fish (1.1%). Twelve isolates were also serotyped by the agglutination method. The most common serotypes detected in the 12 strains tested were 1/4b (75.0%), followed by 1/2a (16.7%), and 1/2b (8.3%). For this study, we used serotyping and detected 6 different virulence-associated genes (inlA, inlB, plcA, plcB, hlyA, and actA) and 16s rRNA using multiplex-PCR. PFGE was performed to determine genetic characterization of L. monocytogenes strains to define the genetic diversity.


Introduction
L. monocytogenes continues to be an important food safety concern [1].The etiological agent of listeriosis is L. monocytogenes, a gram-positive bacterium that contaminates meat, dairy products, unwashed raw vegetables, cabbage, and ready-to-eat food [2]- [4].In immune-compromised individuals, L. monocytogenes causes meningitis, encephalitis, abortion in pregnant women and neonatal infections [5].In the vast majority of human cases, infection is the result of consumption of contaminated food [6].
Typing of L. monocytogenes isolates is another essential element of tracking bacterial contamination sources throughout food systems [7]- [11].However, serotyping method often yields a low power of discrimination in strains [12].
Reliable diagnosis of listeriosis is made by cultural methods, however, the isolation as well as the differentiation of pathogenic and non-pathogenic Listeria remains a time consuming and tedious task [13].The pathogenic potential of Listeria isolates can be assessed by PCR multiple key virulence factors such as hlyA [14], internalin A (inlA), internalin B (inlB) [15], actA, plcA [16], plcB [17].Prf is required for the transcriptional activation of all the virulence genes of the cluster [18].
PFGE has been considered as a standard subtyping method for L. monocytogenes [19].The PFGE method has a high discriminatory power and has been shown to be very accurate and reproducible for fine structure comparison and molecular typing of L. monocytogenes [20] [21].
The purpose of the present study was to investigate the prevalence of L. monocytogenes and examine genetic diversity of the L. monocytogenes isolates from RTE, meat and fish in Seoul, Korea.

Food Samples
We analyzed 1042 food samples taken from different markets in Seoul and obtained from different types of foods such as RTE (kimbap), fish (smoked salmon and seasoned-dried slice fish) and meat (cut raw beef and pork) from 2009 to 2011, were used.

Isolation of L. Monocytogenes
Twenty-five ml of buffer including the swabs were inoculated in 225 ml of Fraser Broth (Becton, Dickinson and company sparks, USA), and blended.All samples were incubated at 30˚C for 48 h.A portion (10 ul) of the enrichment broth was streaked on Palcam agar plate (Merk, Germany).After 24 -48 h incubation at 37˚C the plates were examined for typical L. monocytogenes colonies, which were streaked for purity on horse blood agar plates.Hemolytic colonies on horse blood agar were confirmed as L. monocytogenes by API Listeria kit (Biomerieux, Korea).

Serotyping
The serotypingof L. monocytogenes isolates was performed by a slide agglutination assay using commercially prepared antisera (Listeria antiserum Seikenkit; DenkaSeikenCo, Tokyo, Japan) according to the manufacturer's instruction.

Preparation of Genomic DNA
Genomic DNA was extracted using the AccuPrep ⓡ Genomic DNA Extraction Kit (Bioneer, Korea) according to the manufacturer's instruction.

Identification by the Multiplex-PCR of Virulence-Associated Genes
All of the primers used for specific PCR amplifications of the entire coding sequences of virulence-associated genes are reported in Table 1.PCR was performed in a PCR 9600 thermal cycler (Perkin-Elmer Corporation).A 50-ul aliquot contained buffer (10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl 2 [pH 8.3]), the dNTP mixture (TaKaRa, Japan) 0.25 mM each, 10 pmols of primer, 25 ng of DNA, and 0.8 U of Taq DNA polymerase (Ta-KaRa, Japan).The reaction conditions consisted of template DNA denaturation (94˚C for 3 min), 35 cycles of amplification (each cycle consisted of denaturation at 94˚C for 1 min, annealing at 60˚C for 2 min and elongation at 72˚C for 1min).5 microlitre of the amplified products was separated by electrophoresis in 1.5% agarose gel containing ethidium bromide, and visualized under UV.

PFGE
PFGE was performed according to the PulseNet standardized protocol [22], with ApaI as restriction endonuclease (Roche, Germany).The electrophoretic parameters used were as follows; initial switch time, 4.0 s; final switch time, 40.0 s; sum time, 22 h; angle, 120˚; gradient, 6.0 V/cm; temperature, 14˚C; ramping factor, linear.After electrophoresis, the gels were stained for 15 -20 min in 250 ml of deionized water containing 25 ul of ethidium bromide (10 mg/ml) and destained by three washes of 20 -30 min each using 500 ml of deionized water.
PCR products of the 6 different virulence-associated genes and 16s rRNA (Table 1) were obtained DNA from all Listeria strains considered in this study, the isolates each had 16S rRNA and seven virulence-associated genes (hlyA, plcA, plcB, inlA, inlB, actA, and prfA), suggesting that they are potentially pathogenic.PFGE (ApaI) types of all isolated strains are presented in Figure 1.It demonstrates examples of all PFGE types indicated in our studies.Among the 12, dendrogram analyses of the PFGE profiles showed that the 12 L. monocytogenes isolates from 10 different PFGE profiles with at 80% relative genetic similarity.The most predominant type was A and C.

Discussion
Even though L. monocytogenes causes relatively few cases of human disease in Korea and all over the world, it is still a major problem for public health because it is widespread and present in many animals, food products, raw materials, and the environment.A latest report from the Centers for Disease Control and Prevention (CDC) has estimated 1455 hospitalizations and 255 deaths per year in the United States [1].More specific information of the ecological epidemiology and particularly of the genetic structure of Listeria should help us to understand the origin of listeriosis in the future.
This study was designed to reveal any correlation between L. monocytogenes strains isolated from seven kinds of foods.The strains were analyzed by serotyping, virulence-associated genes, and PFGE.The results of this study showed that the highest contamination rates were meat, fish, and RTE foods, respectively.These findings are in agreement with previous report on outbreak of febrile gastroenteritis in Austria in which its contamination is fish and seafood, raw meat sausages, soft cheese and cooked meat [23].These data are in concordance with the RTE foods observed by Allerberger et al. in a study performed in Germany [24].They found to be recorded an outbreak of 16 cases caused by pre-sliced ready-to-eat (RTE) delicatessen meat (sausage salad) [24].Moreover, evidence from EU-wide routine food safety investigations indicates that a substantial proportion of RTE products may be contaminated by L. monocytogenes [25].The result also confirms RTE, meat, and fish are as one of the important enteropathogens responsible for listeriosis.
In the present study, the L. monocytogenes isolates were serotyped.All of the isolates belonged to serotype 4b, 1/2a, and 1/2b; thus, they also had the epidemic potential.A restricted range of strains was responsible for most of the additional cases at that time, and most human cases are still associated with L. monocytogenes serovars 1/2a, 1/2b and 4b [24] [26].
Our results suggest that three major serotypes may be particularly important as most human infections are reported to be associated with 1/2a, 1/2b, and 4b.Therefore, most of the organisms isolated in this study could lead to human infections and are then possibly pathogenic for humans.
Molecular insight into the genetic characteristics of the 12 L. monocytogenes isolates by determining the presence of virulence-associated genes revealed that isolates carried all the seven traits studied, implying their high pathogenic potential.
In order to define the genetic diversity, we also compared PFGE analyses to determine the genetic characteristics of L. monocytogenes strains isolated from food samples.PFGE is a method with high discriminatory power and it has shown to be very accurate and reproducible for fine structure comparison and molecular typing of L. monocytogenes [12] [20] [21] [27].
Our result showed L. monocytogenes isolates from various different PFGE profiles (10 pulsotypes) at 80% relative genetic similarity.The data from these studies indicate that we have identical pattern from different source with the same 4b serotype.It was shown that RTE (2011-SE-10) unrelated Meat (2011-SE-11) shared identical patterns, suggesting that they were serologically related.These data are in concordance with C pattern.Previous study indicated that the genetic patterns of strains showed no association with any of the properties that correlated with their origin [28].It was found that similar subtypes of strains could be found in different product types and in different processing environments.The results of the present study clearly indicate that the recovery of identical patterns forming various food and patient strains does not prove that a particular food is the vehicle of infection [29].It has been proposed a similar finding with an identical PFGE pattern that was shared by dif-ferent serotypes, even from different flagella antigen groups [30].It was shown that the poor correlation between serotyping and molecular subtyping may be due to horizontal gene transfers or point mutations in genomic DNA resulting in phenotypic shifts that affected serotyping [31].
The results in this study showed specific PFGE types could not be connected with serotype, and especially most fish isolates could differentiate with C, G, and H pattern showing serotypes 1/2a, 1/2b, and 4b which is consistent with results observed in other studies having examined the relation of L. monocytogenes between subtypes and genotypes [32] [33].It has been investigated that correlations between molecular subtyping and serotyping of L. monocytogenes have been reported previously [31].Currently, there is no thorough knowledge of the molecular basis for the relationship between serotypes and molecular subtyping of L. monocytogenes [34].Of interest, it was found that genomic DNA isolates of serotype 4b digested by ApaI enzyme in our study showed relatively distinguishable patterns.This finding is consistent with the study identical PFGE patterns belonged to the same serotype [29] [35].
The application of molecular typing methods for investigating the source of contamination in meat, fish, and RTE foods may lead to a better understanding of the routes of L. monocytogenes spread, thus allowing taking serious measurements to reduce the occurrence of L. monocytogenes in the food production chain.

Figure 1 .
Figure 1.ApaI PFGE restriction patterns for selected L. monocytogenes isolates obtained from RTE, meat and fish in Seoul, Korea.

Table 1 .
Primer pairs used for amplification of virulence genes and 16s rRNA in Listeria isolates.