MOLECULAR DETECTION OF LISTERIA MONOCYTOGENES PATHOGENIC GENES AS FOOD POISONING MICRO-ORGANISM ISOLATED FROM READY- TO- EAT MEALS IN PORT-SAID

Dept. of Biotechnology,

Animal Health Research Institute, Dokki.

Molecular detection of Listeria monocytogenes pathogenic genes as food poisoning micro-organism isolated from ready- to- eatmeals in Port-Said

city markets

(With 3 Tables and 5 Figures)

By

Hanaa A. Ahmed; Amal A. Megahed* and

Gihan M.O. Mohammed*

* Dept. of Bacteriology, Animal Health Research Institute, Port-Said Branch.

(Received at 12/12/2011)

الکشف الجزيئي للجينات مسببة المرض لميکروب الليستيريا مونوسيتوجينز کأحد ميکروبات التسمم الغذائي المعزولة من الوجبات الجاهزة من أسواق مدينة بورسعيد

هناء عبد القادر ، أمل أحمد مجاهد ، جيهان محمد عمر محمد

تم تجميع 105 عينة من الوجبات السريعة (15 عينة لکل من الکفتة، الکبدة، اللانشون، السجق, هوت دوج، البسطرمة وجبنه مطبوخة بالبسطرمة) من المطاعم المختلفة وبائعي الوجبات السريعة بمدينة بورسعيد. وأجري الفحص البکتيريولوجى على العينات حيث أفضت إلي وجود ميکروب الليستيريا مونوسيتوجينز بنسبة 26.6% من عينات البسطرمة فقط بينما کانت جميع العينات الأخرى سلبية لميکروب الليستيريا مونوسيتوجينز. وقد وجد جين الضراوة (بى ار اف ايه) فى جميع المعزولات وهو المسؤل عن بداية إحداث المرض. هذا وقد تمت مناقشة الأهمية الصحية لهذا الميکروب ومدى تأثيره على الصحة العامة وصحة الإنسان وکذلک الشروط الواجب توافرها وإتباعها للمحافظة على سلامة الوجبات السريعة.

SUMMERY

Listeria monocytogenes is a major safety concern for ready- to- eat (RTE) meals, which are frequently contaminated with this pathogen. So a total of 105 random samples represented by 7 kinds of ready –to-eat meals (kofta, fried liver (kibda), luncheon, sausage, hotdog, cooked cheese with basturma and basturma) were collected from different supermarkets and street venders inPort-Said City.L. monocytogenes was isolated from basturma only in 4 samples (26.6%). On other hand L. monocytogenes could not be isolated from any of the other examined samples. Polymerase chain reaction is a powerful technique for detection of pathogens in foods. It is a rapid procedure with both sensitivity and specificity for quick detection and identification of specific pathogenic bacteria from different sources. Listeria monocytogenes detection methods based on PCR amplification of the prfA and hly genes sequences have been reported. The virulence gene prfA was found in all isolates which is responsible for the initiation of pathogenesis so its target is to detect pathogenic L. monocytogenes. The public health hazard of this microorganism as well as recommended measures to improve quality status of ready- to- eat (RTE) meals was discussed.

Key words: Listeria monocytogenes detection, ready- to- eat meals, Food-borne disease, PCR, Public health.

INTRODUCTION

In recent years, there was an increased trend for RTE meals to be consumed both at home and restaurants. RTE meals such as kofta, fried liver (kibda), luncheon, sausage, hot dog, cooked cheese with basturma and basturma have become more popular RTE meals in Middle East countries. They considered the most common RTE sold by street vendors and fast food restaurants (Cressey and Lake, 2005; Vorst et al., 2006; Acciari et al., 2011).

Because of the changing life style in Egypt, people consume a variety of fast foods on a daily basis. Traditional meals- containing fast foods such as kofta, fried liver (kibda) sandwiches became more popular (Hamedy et al., 2007).

Food-borne infections are important public health concern worldwide. According to reports of the World Health Organization and the Center for Disease and Prevention, every year a large number of peoples are affected by food borne diseases due to consumption of contaminated beef (Busani et al., 2005; Cressey and Lake, 2007).

Meatconstitutesthe most important items of human food because of its palatability and high nutritional value, at the same times, meat is prone to contamination from the hands and clothes of staff, knives and other equipments occurred post production or recontamination during further handling(Gill et al., 2001; El-prince and Sayed, 2004).

Listeriosis is a foodborne bacterial disease caused by                  L. monocytogenes, that is widely spread throughout the environment. It had been isolated from soil, water, silage, and many other environmental sources. The organism can grow in PH range of 4.4°C – 9.4°C. resists high salt levels, nitrite, acid and relatively resistant to drying, but easily destroyed by heating and it can grow between 0°C- 45°C. (Janny, 2007; Sue et al., 2009). L. monocytogenes was found in at least 37 species of mammals, both domestic and wild, as well as up to 10% of human may be intestinal carriers. It had been detected in 17 species of birds, some species of fish and shellfish, andis especially pathogenic to high risk populations, such as newborn, pregnant women, elderly, and immunocompromised  individuals (Mugampoza et al., 2011).

Although listeriosis is relatively uncommon, it is a potentially fatal disease. It frequently result in abortions in pregnant women. Even though the symptoms may be relatively mild in the mother, the illness may be transferred to the fetus causing serious illness or fetal death. Some symptoms of L. monocytogenes may include meningitis, encephalitis, septicemia, spontaneous abortion, still birth, and influenza-like symptoms (William and Catherine, 2001; Sutherland et al., 2003; Azevedo et al., 2005; Sue et al., 2009).

Detection of L. monocytogenes by molecular methods is very specific and can be as fast as the immunological assays (Janzten et al., 2006). A number of PCR assays had been described for its detection in foods (Levin, 2003). PCR methods had superior sensitivity when compared to standard nucleic acid probes or immunoassays. However, complex sample preparation methods and the use of gel electrophoresis endpoint detection have hampered the transition of these methods from research to routine use in food microbiology laboratories. Nevertheless, factors influencing the performance of conventional PCR in foods continue to be investigated (Aznar and Alarcón, 2003) and standardized (D’Agostino et al., 2004).

Sequence comparison of individual genes in the prfA virulence gene cluster (pVGC), a central virulence gene cluster, from different Listeria species indicated that priming sites within the genes appeared to be specific for Listeria monocytogenes exclusively. Therefore, the pVGC was targeted for polymerase chain reaction (PCR) assays to detect and specifically identify L. monocytogenes. Each gene of the pVGC was specifically amplified in L. monocytogenes but not in other Listeria species(Jung et al., 2009).

This study was planned to investigate the presence of                    L. monocytogenes among some selected ready- to- eat (RTE) meals using the classical methods of isolation in comparison with molecular tools. It also aimed to detect one of the virulence genes cluster (pVGC) responsible for initiation of pathogenesis (prfA gene).

MATERIALS and METHODS

A - Collection of Samples:

   A total of 105 random samples of RTE meals included fried liver (kibda), kofta, sausage, hotdog, luncheon, cooked cheese with basturma and basturma from different supermarkets and street vender inPort-SaidCity for detection of L. monocytogenes.

B - Isolation:

  Twenty-five grams of each RTE meals samples were homogenized in a stomacher for 2 min in 225 ml of Listeria enrichment broth (Difco), and incubated at 30°C for 48 hr. the samples were divided into 2 portions, one for molecular identification and the other to continue the isolation. After incubation one loopful was subcultured onListeriaOxford medium base. The plates were incubated at 35°C for 24-48 h.

C - Identification:

   Five typical colonies were transferred from Listeria Oxford medium base to Trypticase soy agar with yeast extract for purification. Purified isolates were identified by the Gram- stain, Catalase test, motility test, biochemical tests and Christie- Atkins, Munch- Petersen; test of haemolysis (CAMP Test). Further confirmation of L. monocytogenes the isolates were inoculated in to 10% aqueous stock solution of Manitol, L. Rhamnose and D. Xylose (FDA, 2003).

D - Polymerase chain reaction (PCR)

DNA Extraction: Boiling method (Bansal, 1996). Bacterial pellets were washed once with 1 ml phosphate buffered saline (PBS), pH 7.4, resuspended in a same volume of cold water and incubated in a boiling water bath for 10 min. The clear supernatants obtained after a 5 min centrifugation at 12000g were used for PCR reaction.

Oligonucleotide Primers: in this study 2 sets of primers were used, hyl gene specific for conformation of L. monocytogenes and not any other type of Listeria and prfA gene the initiator gene for pathogenesis. The sequence, cycling conditions and amplicone size were described in table (1). The PCR products were visualized on 1.3% agarose gel in 1x TBE using GeneRuler 100 bp plus DNA Ladder (Fermentas Cat. No. #SM0323).

Table 1: Shows the sequence, cycling conditions and amplicone size of the used genes:

RESULTS

In the present study, all types of RTE food examined (105 samples listed in Table 2 and Fig. 1) were negative to the presence of Listeria except that of Basturma, four samples out of the examined fifteen were positive by isolation with an incidence rate of 26.6% and overall recovery rate of 3.8%. All Listeriaspp. hydrolyze esculin and the inclusion of esculin and ferric iron in enrichment or plating media results in the formation of an intense black color (Fraser and Sperber, 1988). This is due to the complexation of the ferric iron with 6, 7-dihidroxycoumarin, the product of esculin cleavage by β-D-glycosidase, resulting in a black precipitate.

Table 2: Incidence of L. monocytogenes in the examined samples of RTE meals.

Fig. 1: Incidence of L. monocytogenes in the examined samples of RTE meals.

These isolates were subjected to hemolysis, Christie- Atkins, Munch- Petersen; test of haemolysis (CAMPTest) and acid production tests for differentiation of listeria species as listed in Table 3 and Fig 2. An “arrowhead” hemolysis is seen between growth of Listeria monocytogenes and Group B Staph aureus. This is because of alpha toxin produced by Staph aureus interacts with Christie- Atkins, Munch- Petersen; test of haemolysis (CAMP Test) factor and produce synergistic hemolysis.

Fig. 2: Listeria monocytogenes on blood agar Christie- Atkins, Munch- Petersen; test of haemolysis (CAMP Test) showing the characteristic arrowhead hemolysis between basturma isolates only and Staph aureus.

Table 3: Biochemical differentiation of Listeria species in the examined isolates:

+:positive, +/–: weakly positive, –: negative, CAMP: Christie- Atkins, Munch- Petersen; test of  haemolysis, V: variable

         C= control,  T = tested isolates

All samples were subjected to PCR after primary enrichment, The PCR results didn’t give any results after first PCR run from initial culture (Fig. 3) compared to L. monocytogenes reference strain; by re- PCR only basturma gave a characteristic band at 702 bp to hyl gene specific for L. monocytogenes (Fig. 4).

The positive PCR samples for hyl gene were examined for the presence of prfA gene and gave the characteristic band at 1060 bp compared to reference strain (Fig 5) also at the 2^nd round PCR (Re- PCR). 

DISCUSSION

The microbiological safety of food products is of extreme importance to industry, public health officials, regulatory agencies and the public little information is available regarding the occurrence of    L. monocytogenes in meat, meat product and soft cheese in retail market in Port-Said City. This survey provided a clear picture for the prevalence of L. monocytogenes in RTE meals offered to consumers.                           L. monocytogenes was isolated from 4 basturma samples with a percentage of (26.6%) in Table 2 and figure 3. In this concern, isolation of L. monocytogenes in final products of basturma were have been reported by Steven et al.)2006).

An additional microbiological hazard of potential concern in basturma is Listeria monocytogenes. Basturma is sold as a raw product and some people may consume basturma as it is, while cooking by the consumer is important for preventing food borne illness due to listeriosis associated with this product. L. monocytogenes growth on the finishing product is unlikely because of its reduced water activity (Aw)^. Guidance from USDA states that, decreasing a products water activity to < 0.92 and PH <4.39 will preventL. monocytogenes growth (USDA, 2004). The American Meat Institute Foundation (2005) stated thatunless the finishing basturma (contained L. monocytogenes at levels greater than 4 log CFU, or supported growth to such a level), cooking procedures should dramatically decrease the risk of basturma -linked Listeriosis. In general, the organism has been found in raw milk, soft cheese, fresh and frozen meat and vegetable products (Hege et al., 2000)

The ability of food borne micro-organisms asL. monocytogenes to become disseminated from naturally contaminated foodsto various  hand and food contact surface is well know.The results achieved from Tables 2and 3 revealed that, L. monocytogenes could not be detected in fried liver (kibda), kofta, Luncheon,cooked cheese with basturma, sausage or hot dog samples. The incidence of L.monocytogenes in luncheon meat was Lower than that obtained by Gombas et al. (2003) who found it in 0.89% of luncheon meat. In contrast, L. monocytogenes could not be isolated from meat samples (Elgazzar and Sallam, 1997; Mohamed; Ali, 1999; Saad et al., 2001). This wide variation may be explained in terms of geographic location, isolation methods and kinds of media employed (Akpolat et al., 2004). Cross-contamination, which can occur within environment of food-processing equipment, is considered to be a possible source of Listeria contamination in processed meat such as luncheon. L. monocytogenes is able to attach to and survive on various working contact surfaces (Borucki et al., 2003). One reason may be its ability to form biofilms (Wong, 1998). Furthermore, during further transformation processes of raw meat into meat products L.monocytogenes can be introduced, where the amount depends on the extent of cross-contamination, personal and general hygienic measures and the process parameters (Glass and Doyle, 1989). In addition, minced/chopped meat products as luncheon, by their nature, undergo extensive processing and handling during their production. This leads to greater opportunities for L. monocytogenes contamination (Tompkin     et al., 1992 and Uyttendaele, 1997). The difference occurring, either increase or decrease in the incidence of other literatures than that obtained results, this attributed to the nature processing load to RTE meals, also due to the level of contamination of other additives, which added to the RTE meals and difference in country and type of sample analysis.

The mortality rate of listeriosis is very high, approximately 30% (Griffiths, 1989), and for this reason the Food and Drug Administration maintains a policy of zero-tolerance for L. monocytogenes (Anon, 2003). With this concern, the ability to detect this pathogen at low levels is considered essential because most of the foods susceptible to contamination are ready-to-eat products, which are not cooked or otherwise processed before consumption. These considerations underline the benefit of monitoring sensitive L. monocytogenes to ensure the microbiological quality of foods and to reduce risks for public health.

The smoking, cooking, fermentation or drying processes used to make the samples tested can be considered effective antimicrobial processes for control of L. monocytogenes on RTE meals)Steven et al., 2006). The European community directive on milk and milk-based products specifies zero tolerance for soft cheese and the absence of the organism in 1 g of the product (Food Safety and Inspection Service, 2000).

Current microbiological culture methods rely on growth in culture media, followed by isolation, and biochemical and serological identification. However, the detection of this pathogen in food by these standard culture methods is made difficult by the sporadic or low levels of contamination (<100 cfu/ g) can't be relied incase of the presence of a high level of background micro-flora and competitor organisms that could mask the presence of L. monocytogenes, or even interference due to food matrix components (Norton et al., 2001). Moreover, these methods are laborious and time consuming, requiring a minimum of five days to recognize Listeria spp. and about 10 days to identify                  L. monocytogenes by confirmatory tests (Anon, 1996), while immediate action should be taken in case of contamination since it is of fundamental importance to ensure the safety of food products, especially the case of those food matrices having short shelf-lives, such as meat or dairy products.

In the past years, advancements in biotechnology have resulted in the development of rapid methods that reduce analysis time and offer great sensitivity and specificity in the detection of pathogens. Among these, PCR has been increasingly used for the rapid, sensitive and specific detection of foodborne pathogens (Olsen et al., 1995). However, the successful application of PCR assays to food samples has been hindered by the lack of a rapid and efficient method for the preparation of PCR-amplifiable DNA (Lantz et al., 1994). The presence of PCR inhibitors in food samples (Rossen et al., 1992; Bickley et al., 1996) represented the main limitation in this kind of assays due to the production of false negative results. For this reason, the application of PCR-based methods is closely linked to the selection of suitable methods for DNA extraction.

The results obtained from isolation were in line with that of molecular diagnosis as PCR detected only the presence of                      L. monocytogenes in 4 basturma samples at 2^nd PCR. This clarify the reason why PCR didn’t give any result at first time, but gave results with Re-PCR during the detection of hyl and prfA genes.

Gouws and Liedemann (2005) and Amagliani et al. (2007) associated the main limitation with PCR application to food-contaminating microorganisms concerns to the presence of inhibitory substances that are coextracted with DNA and may be present in the sample, causing a failure in the amplification reaction which leads to false negative results. Therefore, quality and purity of extracted nucleic acids are the main requirements for a PCR-based detection assay at the mean while the selection of a proper extraction method is determinant for a successful and valid PCR analysis.

This consideration could explain the results obtained for samples extracted using the boiling method suggested by Bansal et al. (1996), showing faint signals in gel analysis. Although lysis by boiling appeared to be the most convenient extraction protocol, particular care should be taken when food contamination with very low bacterial count is suspected.  Cooray et al. (1994) noted that boiling for 5 min at 100°C was superior to heating at 95°Cfor releasing DNA from cells.

Oligonucleotide primers targeted to the L. monocytogenes hlyA gene were highly species specific and provide a means for easily differentiating L. monocytogenes from other hemolytic species of Listeria (Deneer and Boychuk, 1991). Several genes coding for various aspects of virulence in L. monocytogenes had been identified and used for PCR detection of the organism. Wernars et al. (1992) made use of the prfA gene to develop a highly specific PCR for recognition of pathogenic L. monocytogenes strains. The primers used, prfA-A/ prfA-B, flank a 1060-bp sequence encompassing the entire prfA gene directed against nucleotides 181 to 207 and 1462–1482 of the prfA gene sequence, were found to be specific for all virulent strains of                 L. monocytogenes tested, and readily distinguished them from a-virulent strains of L. monocytogenes and representatives of the other six Listeria species (Levin, 2010).

In conclusion, this study has demonstrated the presence and distribution of L. monocytogenes in a variety of RTE meat products in Port-Said. PCR was more rapid and sensitive method, useful for the rapid detection of L. monocytogenes in a reasonable period of time which may avoid costly recalls and reducing outbreaks. As well as any ready-to-eat foods such as hot dogs, luncheon meats, cold cuts, fermented and dry sausage should be eaten only after they have been reheated to a high temperature.

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