Detection and identification of Salmonella species in minced beef and chicken meats by using Multiplex PCR in Assiut city

The present study was undertaken to determine the incidence and distribution of Salmonella species in selected meat and chicken products purchased from retail supermarkets in Assiut, Egypt. A total of 75 samples including 25 samples each of minced frozen beef, frozen chicken legs and frozen chicken fillets were collected over a 7-month period between January and July 2009 and examined for the presence of Salmonella species. In addition, 28 children stool cultures were collected from hospitalized children resident in Pediatric University Hospital with diarrhea or fever. Out of the total 75 meat samples examined, Salmonella was detected in 5 (20%) of minced frozen beef, 9 (36%) of frozen chicken leg and 13 (52%) of frozen chicken fillet samples analyzed. Regarding the examined 28 children stool cultures, 3 (10.71 %) were found Salmonella positive. Of the total 30 Salmonella positive samples from all examined samples, five selected Salmonella isolates were further identified using multiplex PCR (m-PCR). Two serovars were the dominant serovar identified was Salmonella entrica subsp. entrica serovar Enteritidis (2 chicken leg isolates and 2 chicken breast fillets) followed by Salmonella entrica subsp. entrica serovar Kentucky (one minced beef isolate). The public health hazards of Salmonella were discussed and the suggestive measures to protect the consumers and improve the quality of meat and chicken products were given.

Introduction associated with poor sanitation and are primarily diseases Food-borne illness is a major international health of developing countries (15). problem (1,2). Each year, millions of persons become ill In humans, Salmonella are the cause of two from food-borne diseases, though many cases are not diseases called salmonellosis: enteric fever (typhoid), reported (3).
resulting from bacterial invasion of the bloodstream, and Salmonella is one of the most important pathogenic acute gastroenteritis, resulting from a food-borne genera implicated in food-borne bacterial outbreaks and infection/intoxication (16). diseases (1,4). Salmonella infections are worldwide and Outbreaks have been associated with a wide variety constitute an important public health problem in many of foods. Dominant serotypes from clinical cases vary parts of the world (5,6). There are several transmission with geographical region: for example, S. enteritidis is routes for salmonellosis, but the majority of human the most common in Europe, while S. typhimurium is the infections are derived from the consumption of most common in Oceania (17). contaminated foods especially those of animal origin (7).
In many countries, poultry meat products continue A variety of food products, especially poultry and other to be a major cause of human enteritis (18,19) and types of meat products including minced meats, are the outbreaks are reported regularly involving Salmonella most important sources of human Salmonella infection species (20,21). Moreover, Salmonella is the most (8,9,10,11). International outbreaks caused by a range of common food poisoning bacteria associated with foodstuffs contaminated with different Salmonella refrigerated poultry (22). serotypes have been reported (12,13,14). Most serotypes The detection and identification of Salmonella spp. of S. enterica cause self-limiting gastroenteritis is time consuming to the food industry (23). To detect characterized by diarrhoea, abdominal cramps and Salmonellae more rapidly, an alternative method to the sometimes vomiting and fever. Two serotypes, S. typhi conventional culture method was evaluated using polymerase chain reaction (PCR). PCR has been and S. paratyphi, causes systemic fevers that can be fatal demonstrated to be a very specific and sensitive method sterile lactose broth. The homogenate was incubated at for the detection of Salmonellae (24). In the last decade, 37°C for 24 h. there has been a wide interest in the use of the multiplex Selective Enrichment: 0.1 ml and 1 ml of the PCR (mPCR) technique. mPCR approaches have been incubated pre-enrichment homogenate were transferred largely applied to detect different species of several to 10 ml Rappaport-Vassiliadis broth (RV) (Oxoid) and microbial niches, to differentiate closely related species Selenite cystine (SC) broth (Difco) as selective and to recognize single species (25). enrichment, respectively. RV broth incubated at 42°C for Therefore, the goal of this study was to determine 24 h and SC broth incubated at 37°C for 24 h. the incidence of Salmonella spp. in minced beef, chicken Selective Plating: At the end of the incubation period, meats and human in Assiut (Egypt) and to use multiplex a loopful from each of the selective enrichment broths PCR (m-PCR) for their identification.
was streaked onto Salmonella-Shigella (SS) agar (BBL), and incubated at 37°C for 24 h. The plates were examined

Material and Methods
for the presence of typical colonies of Salmonella (transparent colonies with black centers on SS agar). Collection of samples: A total of 75 random samples Confirmation: Smears of suspected colonies were of meat and chicken products (25 samples each of minced stained with Gram's stain and examined morphologically frozen beef, frozen chicken legs, and frozen chicken for staining characters. Presumptive Salmonella colonies breast fillets without skin) were collected from different were then subjected to initial screening tests using triple retail supermarkets and groceries in Assiut city. The sugar iron agar (TSI) slant, lysine iron agar (LIA) slant minced frozen beef samples were collected in (Merck), urea broth (Merck) and lysine decarboxylase presterilized plastic bags while, frozen chicken meat broth (Oxoid). TSA incubated at 37°C for 24 h and LIA samples obtained in their casing as sold to the consumers. incubated at 37°C for 24-48 h. All biochemical tests were The collected samples were transferred directly to the performed at 37°C for 18-24 hrs including citrate laboratory in an ice box with a minimum of delay, where utilization, indol production, methyl red, motility, urease they were prepared for bacteriological examination.
and Voges-Proskauer (27). Preparation of samples: At the laboratory, frozen DNA extraction for multiplex PCR assay samples were thawed by overnight refrigeration. Each DNA was extracted from selected pure cultures of sample was aseptically and carefully freed from its bacteria that had been enriched at 37°C for 18 h in brain casings and mixed thoroughly in sterile mortar.
heart infusion (BHI) broth. One ml of the BHI broth was Human samples: To identify the occurrence of collected in an eppendorf tube, centrifuged at 15000 rpm Salmonella infections in hospitalized children in Assiut, for 5 min and the supernatant discarded. The cell pellets the cases of study was conducted. The cases were defined were resuspended in 1 ml phosphate buffered saline as the children, resident in Pediatric Uni. Hospital; Assiut (PBS, Oxoid), centrifuged at 15000 rpm for 5 min and the Uni., with diarrhea or fever between January and July supernatant discarded. DNA extractions were performed 2009. A stool culture examined for Salmonella. The on the cell pellet using the DNeasy Blood and Tissue kit parents of the cases were interviewed, by a single (Qiagen, Hilden, Germany) according to manufacturer's investigator using a standardized questionnaire instructions. addressing the family's consumption of, and purchasing For the multiplex PCR, four primer pairs were and preparation conditions for, various foods such as used. All of the primer sequences are shown in Table 1. poultry and beef, and their contacts with people having Multiplex PCR reactions were performed with Techne presented with an episode of diarrhea.
Cyclogene Thermocycler using a total volume of 25 µl, Isolation of Salmonella spp. (26) which was contained within a 0.5 ml thin-walled PCR Pre-enrichment: Twenty five grams of the examined tube. The optimal amplification reaction mixture samples were weighed aseptically into sterile blender contained 12.5 µl 2 × QIAGEN Multiplex PCR Kit container and thoroughly homogenized with 225 ml of  indicates the absence of the bacteria, but this result may The PCR protocol consisted of the following steps: be due to low sensitivity and specificity of the method (i) an initial denaturation step of 2 min at 95°C; (ii) 30 used in isolation. cycles, with 1 cycle consisting of 1 min at 95°C, 1 min at In this study, the identical strain was identified 57°C, and 1 min at 72°C; and (iii) a final elongation step using m-PCR as Salmonella enterica subsp. enterica of 10 min at 72°C. The PCR products were serovar Kentucky (S. Kentucky). electrophoresed in 1% (wt/vol) D-1 agarose (Pronadisa, Salmonella enteritidis has become the Madrid, Spain), stained with 2 µg of ethidium bromide predominant strain that causing food poisoning. (Sigma-Aldrich, Madrid, Spain) per ml, and photo-Investigation of Salmonella enteritidis outbreaks indicate graphed under UV light. In each PCR run, a nontemplate that its emergence is largely related to consumption of control was included to detect possible external DNA poultry or eggs (44). contamination.

Primer Length(nucleotides) Primer sequence Amplicon product (bp) References
Food-borne salmonellosis often follows consumption of contaminated animal products, which

Results and Discussion
usually results from infected animals used in food The obtained results of this study are summarized production or from contamination of the carcasses or in Tables 2 & 3 and Figure 1. edible organs (45,46).

Screening of minced frozen meats for Salmonella
The true incidence of salmonellosis both in humans Out of 25 minced frozen meat samples examined, and animals is difficult to evaluate because of lack of an five samples (20.0%) were found to be contaminated epidemiological surveillance system in place, which is with Salmonella (Table 2). Previous studies conducted in particularly true in developing countries. However, in Assiut, Egypt, indicated the occurrence of Salmonella in countries with a reporting system, the number of different food animals, meat and meat products outbreaks particularly in humans has increased (30,31,32). Other investigators could recover the considerably in recent years (47,48). Carrier states of organisms from minced meat samples in variable humans are of concern to the food manufacturing and percentages such as (30) (5%), (33) (12%), (34) (5%), food service industries because of the perceived risk of (35) (1.5%), (36) (11.4%), (37) (6.3%), (38) (12.1%) and contamination of food by infected food handlers and the (32) (6%). Summarized data from several European risk of food-borne disease outbreaks (49). Meat countries showed that Salmonella prevalence in minced processing and packaging at the wholesale or retail levels beef ranged from 0.0% to 3.6%, with a mean of 1.1% are likely to contribute to the higher levels of (39).
contamination in minced beef compared to beef Contradictory report had been published on the carcasses. The presence of even small numbers of Salmonella in carcass meat and edible offals may lead to absence of Salmonella in the examined 30 minced meat heavy contamination of minced meat and sausage. When samples collected from different localities in Assiut city meat is cut into pieces, more microorganisms are added to poultry carcasses and products, together with the the surfaces of exposed tissue. Raw meats, particularly consumption of undercooked poultry meat (53). minced meats have very high total counts of Varying incidence rates of Salmonella in chicken microorganisms and Salmonellae are likely to be present and/or chicken parts were reported. (54) found in large numbers (30). Salmonella in 26.3% of fresh whole chickens, 26.7% of breasts, 14.3% of legs, 0% of drumsticks, 0% of thighs, During conventional slaughter procedures and and 40% of wings. (55) reported Salmonella contamfurther processing to prepare poultry and meats for ination around 40% for each of wings, legs and giblets. consumption, microorganisms are introduced into and Moreover, Straver et al. (56) pointed out that nineteen onto carcasses (50). Prevention of contamination during fillets (8.6%) were contaminated with Salmonella on slaughtering and subsequent processing has therefore chicken breast fillet (chilled raw fillets without skin) been identified as by far the most important factor in collected from five local retail outlets in The Netherlands. safeguarding the microbiological quality of poultry (51).

Detection and identification of Salmonella species in minced beef and chicken meats
Also, poultry meat was extensively contaminated Several gastroenteritis outbreaks have been reported in with Salmonella (40%) in Sao Paulo, Brazil (57). Jordan in the pervious years, many of which were owing Contradictory report has been published on the absence to S. enterica as a causative agent as a result of of Salmonella from the examined 30 chicken quarter consumption of contaminated food-products (1).
samples collected from different localities in Assiut city

Screening of chicken meats for Salmonella
(31). The findings outlined in Table (2) showed that nine Compared to the results of these studies, incidence samples (36.00%) of frozen chicken legs and thirteen that we have obtained appears to be quite high. The samples (52.00%) of frozen chicken breast fillets were Salmonella strains detected in our survey were isolated contaminated with Salmonella. from chicken samples purchased from different Chicken products are widely acknowledged to be a supermarkets. Some earlier reports (58,55) indicated significant reservoir for Salmonella. They have lower percentage of Salmonella in chickens bought from frequently been incriminated as a source of Salmonella butchers' and poultries' shops compared to supermarkets. contamination and consequently thought to be major It is possible that the chicken parts may be contaminated sources of the pathogen in humans (52,9). Furthermore, by Salmonella at multiple steps such as production, one of the commonest causes of Salmonella infection processing and distribution until retail marketing. reported in humans has been through the handling of raw Detection and identification of Salmonella species in minced beef and chicken meats Chicken packaging may be a potential vehicle for using multiplex PCR, four primer sets were used in the introduction of pathogens in retail and in particular for same reaction mixture (Table 1). Analyzing the PCR profiles, 4 out of Salmonella isolated strains showed one the cross-contamination of Salmonella (58).
amplified product (293-bp), (Fig. 1) that is specific for In the current study, the identical strains were serovar Enteritidis and one strain showed amplification identified using m-PCR as Salmonella enterica subsp.
product at (183-bp) that is specific for serovar Kentucky. enterica serovar entertidis (S. entertidis). The isolation In the current study, the identical strains were identified of S. entertidis from chicken carcasses and/or chicken using m-PCR as Salmonella enterica subsp. enterica parts has been reported by some other researchers serovar Entertidis (S. entertidis) and Salmonella enterica (59,60,61). Worldwide, S. entertidis is a clinically subsp. enterica serovar Kentucky (S. kentucky). prevalent Salmonella serovar, which is associated with From the results of the present study, it could be the consumption of foods containing eggs or poultry meat concluded that Salmonella is widespread in minced from systemically infected chickens (60). In Finland, S.
frozen beef, frozen chicken legs and fillets samples entertidis has been the most common serovar in poultry obtained from retail supermarkets in Assiut, Egypt. It and humans (59).
could be a potential vehicle for food-borne infections and The identification of S. enteritidis and/or S.
implementation of preventive measures and consumer kentucky in meats was applied on DNA isolated from the food safety education efforts are needed. Proper cooking selected pure cultures of bacteria; because Salmonella is of meat and chicken products before consumption and not uniformly distributed throughout contaminated improving personal and meat hygiene in the line of meat samples (62). Consequently, it is essential to increase the production from farm to fork should be adopted to ensure processed quantity of the sample to enhance the the safety of meat and meat products for human probability of detecting such bacterium and to avoid consumption. sampling errors.
This finding recommends that PCR could be used

References
to gain a quick and a reliable idea about the status of the