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Praca oryginalna Original paper
Listeria monocytogenes is a ubiquitous Gram-posi-tive intracellular pathogen isolated from food and soil, and it is especially common in fresh foods (10, 13). As a psychrotrophic organism, L. monocytogenes is able to survive in food products that are kept at a refri-gerated temperature (4°C) and high salt concentration (10%). Although the minimal infective dose or accep-table limit of L. monocytogenes in ready-to-eat (RTE) foods may be different in different countries (10), according to EU regulation 2073/2005 in such food the limit is 100 cfu/g. The Hly A gene, i.e. one of the encodes listeriolysin-O (LLO), can be measured to determine the pathogenic risk of L. monocytogenes, which survives in the macrophage and neutrophile cells in the host (9).
Since RTE foods are commonly consumed in hospi-tals and restaurant, they are high risk for the public (23). During cutting, grinding, and other processing steps, the structure of vegetables is broken and some liquid, is released, which serves as a good culture
medium for microorganisms (7, 17). As a result of poor handling, an increase in microorganism growth may cause a rapid decay of vegetables and reduce their shelf life (20). Furthermore, vegetables used to prepare traditional ready-to-eat foods are watered from the Asi River, which is polluted by cities, factories and animal waste, and may carry L. monocytogenes from Lebanon, Syria and Turkey. Therefore, those vegetables are at risk of contamination with L. monocytogenes because of their exposure to polluted water, improper handling and broken cold chain (19). Several studies reported that cabbage, lettuce, parsley, and tomato were the main sources of L. monocytogenes (3, 11).
Abagannus (eggplant salad, ingredients: eggplant, garlic, parsley, pomegranate juice, roasted pepper, salt, and tomato), hummus (ingredients: chickpeas, cumin, garlic, lemon juice, olive oil, red pepper, and salt), walnuts with red pepper (ingredients: cumin, dried raw onion, hot red pepper, salt, and walnuts), parsley salad (ingredients: lemon, mint, olive oil, parsley, red
Detection of Listeria monocytogenes
by using PCR method in refrigerated ready-to-eat
food consumed in Turkey
MEHMET ELMALÝ, SULEYMAN ÖNER, HILMI YAMAN*
Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Mustafa Kemal University, TR-31040 Antakya/Hatay, Turkey
*Department of Nutrition and Dietetics, Aydýn School of Health Sciences, Adnan Menderes University, TR-09100 Aydýn, Turkey
Elmali M., Öner S., Yaman H.
Detection of Listeria monocytogenes by using PCR method in refrigerated ready-to-eat food consumed in Turkey
Summary
This study aimed to detect the prevalence, risk and importance of Listeria monocytogenes in ready-to-eat (RTE), traditional food products, using PCR in the Antakya/Hatay district in the southeast of Turkey, which is a popular tourist destination. L. monocytogenes was isolated and identified from 2.09% of the 239 RTE food samples tested. L. monocytogenes was found in 3 (2.58%) of 116 salad samples, and 2 (6.66%) of 30 abagannus samples. However, no L. monocytogenes contamination was detected in the samples of broad bean paste, hummus (crushed chickpeas), parsley salads, traditional salted yoghurt, thyme salads, and walnuts with red pepper. In conclusion, our study showed a relatively high risk of L. monocytogenes in some traditional RTE foods in the Antakya/Hatay district of Turkey.
The observed L. monocytogenes contamination may have been caused by a high microbial load of the ingredients, cross-contamination, poor production and processing conditions, incorrect handling, or watering vegetables with polluted water.
Med. Weter. 2013, 69 (1) 37
pepper, and vinegar), thyme salad (ingredients: olive oil, onion, thyme, parsley, pepper, and pomegranate juice), broad bean paste (ingredients: cumin, garlic, mashed broad bean, olive oil, parsley, pepper powder, red pepper, and sesame powder), salted yoghurt (ingredients: goats milk (10 parts), cows milk (0.2--0.5 parts), red pepper and olive oil), and fresh-made salads are traditionally important RTE foods, especially in the Antakya/Hatay district in Turkey.
This is the first study involving the isolation and identification of L. monocytogenes in ready-to-eat foods in a tourist area of the Antakya/Hatay district in Turkey to detect the prevalence of L. monocytogenes.
Material and methods
Samples. A total of 239 ready-to-eat food samples were collected throughout July, August and September 2011, mainly from traditional restaurants in the Antakya/Hatay district. The samples were transported under refrigerated conditions (4°C) to the Food Hygiene and Technology Department of Mustafa Kemal University, Veterinary Medicine Faculty, and analysed.
Reference strains. L. monocytogenes ATCC 19111, S. aureus and Rhodococcus equi ATCC 33701 cultures were used in this study.
Isolation and identification. Each 25 gram food sample was added to 225 ml ONE broth-Listeria medium and incubated at 30°C for 26 hours. After incubation, the samples were homogenised with a stomacher for one minute. Subsequently, 10 ml of homogenate was cultured by the Brilliance Listeria plate stain technique at 37°C for 26 hours. The Listeria rapid test (Oxoid) was performed for blue/green colonies to confirm their identity as Listeria spp. (19). CAMP (S. aureus-R. equi), D-mannitol, rhamnose, xylose and nitrate reduction tests were performed for the Listeria spp. positive colonies for species identification (13). Finally, the colonies identified as L. monocytogenes were confirmed by PCR.
Polymerase chain reaction (PCR). A kit (Vivantis, Malaysia) was used to extract total DNA from 200 µl volumes of salads and traditional food samples. A 388-bp conserved region of the HlyA LLO-liste-riolysin O gene of L. monocytogenes was amplified by PCR with 5-GAA TGT AAA CTT CGG CGC AAT CAG-3 and 5-GCC GTC GAT GAT TTG AAC TTC ATC-3 sense and antisense primers, respectively (4, 5). PCR reactions were performed in a thermal cycler (Techne 3000G, Barloworld Scientific, UK). A Qiagen Fast Cycling PCR kit (Qiagen, Germany) was used as recommended by the manufacturer. PCR was performed under the following reaction conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles of denatura-tion (96°C for 5 sec), annealing (61°C for 5 sec) and extension (68°C for 9 sec). The reaction was termi-nated after a final hold at 72°C for 1 min. The final specific PCR products were visualised by 1.5% agarose gel electrophoresis. The gels were examined for specific size bands with a UV transilluminator.
Statistical analysis. The frequency and percentage of contaminated items were calculated to determine L. mono-cytogenes distribution.
Results and discussion
L. monocytogenes was isolated and identified from 2.09% of the total of 239 RTE food samples. L. mono-cytogenes was found in one salad sample in July and in two salad samples in August (2.58%) out of the total of 116 salad samples, as well as in one abagannus sample in July, and one abagannus sample in August (6.66%) out of the total of 30 abagannus samples. No L. monocytogenes was found in salad or abagannus samples in September. Likewise, no L. monocytogenes was detected in broad bean paste, hummus, parsley salads, traditional salted yoghurt, thyme salad, and walnuts with red pepper. The data on the presence of L. monocytogenes in food samples are displayed in Fig. 1 and Tab. 1.
Fig. 1. PCR amplification of L. monocytogenes. Line L: 100 bp DNA ladder (New England BioLabs); Lines 1 to 5: positive food samples; Line 6: negative control; Line 7: positive control (standard L. mono-cytogenes ATCC 19111)
Tab. 1. Prevalence of L. monocytogenes in RTE food
)r e b m u n ( s e l p m a s d o o F Numbsearmopflepossiitve pLos.imitvoenoscaymtopgleesne(%s) ) 6 1 ( s u m m u H 0 0 ) 6 1 1 ( d a l a S 3 2.58% ) 5 1 ( r e p p e p d e r h ti w s t u n l a W 0 0 ) 2 2 ( s d a l a s y e l s r a P 0 0 ) 0 3 ( s u n n a g u b A 2 6.66% ) 4 1 ( s d a l a s e m y h T 0 0 ) 1 1 ( e t s a p n a e b d a o r B 0 0 ) 5 1 ( tr u h g o y d e tl a s l a n o it i d a r T 0 0 ) 9 3 2 ( l a t o T 5 2.09%
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Fresh vegetables are an important part of the human diet, especially considering the increasing incidence of obesity and coronary artery diseases in industriali-sed countries. Vegetables are recommended because of their low calorie contents and fast and convenient serving systems. However, as a result of improper pre-servation and storage conditions as well as the natural properties of chopped vegetables, RTE foods made from vegetables have short shelf lives and often pose a high risk for human health.
Several studies have been devoted to the occurrence of L. monocytogenes in several RTE foods in different countries; for example, Pianetti et al. (20) and Copo-nigro et al. (8) reported that no L. monocytogenes were found in 56 and 1158 ready-to-eat vegetable salad samples from Italy, respectively. In another study, Loncarevic et al. (16) reported that L. monocytogenes was isolated from 2 out of 179 lettuce samples in Norway. Hossein et al. (14) reported that L. mono-cytogenes was isolated from 1 out of 71 vegetable samples in Trinidad. Similarly, Jalali and Abedi (15) found L. monocytogenes in 1.6% of 617 RTE food samples. Pingulkar et al. (21) detected 7 strains of L. monocytogenes in 105 isolates from 66 samples containing 13 different ingredients, whereas 30 strains of L. innocua were detected. Higher numbers of L. mono-cytogenes were detected in this study compared to those of Coponigro et al. (8), Hossein et al. (14), Loncarevic et al. (16), Pianetti et al. (20), Pingulkar et al. (21), Rodriguez et al. (23), and Santos et al. (24). These differences may have resulted from the low number of samples analysed, the initial contamination level, or improper food production, handling and service techniques.
Bouayad and Hamdi (6) found Listeria spp. in 21 out of 227 (9.3%) RTE food samples, 6 of which (2.6%) were identified as L. monocytogenes. Ponniah et al. (22) reported L. monocytogenes in 102 out of 306 (33%) vegetable samples. Awaisheh (2) reported that L. monocytogenes was isolated from 19 out of 360 RTE food samples in Jordan. Likewise, Vasilev et al. (25) found L. monocytogenes in 9.2% of 3712 salad samples in Israel in 1998-2007. Gelbicova and Karpiskova (12) reported that L. monocytogenes was isolated in 55 out of 2180 RTE foot samples (2.5%) in the Czech Republic in 2004-2008. Arumugaswamy et al. (1) reported that L. monocytogenes was found in 5 out of 22 (22%) leafy vegetable samples. The rates of contamination are all significantly higher than those found in our study. This may have been caused by the initial microbiological load in ingredients, production and preservation conditions, cross-contamination, or handling practices. The Asi River runs through both Syria and Turkey, and is used for irrigation and as a water supply for some residential areas in the region. The Asi River is intensively used for agricultural activities in the region. Therefore, sewage sludge, fertiliser, as well as industrial and animal waste
present in the Asi River may constitute a risk for public health.
The results of the above studies indicate that the Asi River carries many different types of microorganisms that may contaminate farmed vegetables used as main ingredients in RTE foods, and thus pose a high risk for human health as a result of improper food production, storage and consumption, especially in the case of RTE food.
Conclusion
The results of our studies show that there is a relati-vely high risk from L. monocytogenes in traditional RTE foods in the Antakya/Hatay district, which is a common tourist destination. There is a possibility of contamination resulting from the watering of vegeta-bles with polluted water and subsequent cross-conta-mination from water to raw vegetables used in RTE foods. Further studies to investigate contamination sources and epidemiological studies are recommended. In the future, preventive measures should be taken against L. monocytogenes in RTE foods in terms of public health, particularly for pregnant women, children and people with immunosuppressive condi-tions. Finally, HACCP and GMP plans, including Listeria spp. criteria in RTE foods, must be imple-mented so that these food products can be exported throughout the EU.
Acknowledgements
We would like to thank Assoc. Prof. Dr. V. Soydal Ataseven for allowing us to use his laboratory facili-ties.
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Corresponding author: Assoc. Prof. Dr. Mehmet ELMALI, Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Mustafa Kemal University, Tayfur Ata Sokmen Campus, 31040 Antakya, Hatay, Turkey; e-mail: elmali25erz@gmail.com
