1. Introduction

Everyone faces the risk of contracting a foodborne illness simply because everyone eats. The World Health Organization (WHO) defines ‘Food Safety’ as the assurance that, food will not cause harm to the consumer when prepared and/or eaten in accordance with its intended use. Furthermore ‘Food Hygiene’ is defined, as all the measures necessary to ensure the safety, soundness, and wholesomeness of food at all stages of its production or manufacture until its final consumption [1].

Food industries worldwide have to conform to microbial standards associated with the safety and the quality of their products. Safety aspects are of major importance, thus are determined clearly and unambiguously. Usually, strict limits up to no tolerance are implemented for pathogenic microorganisms that could cause severe health problems to consumers [2].

Over the last 25 years, the global incidence of foodborne infections has markedly increased, with nearly a quarter of the population at a high risk of illness [3]. The World Health Organization [1] estimates that foodborne and waterborne diarrheal diseases together kill around 2.2 million people annually. According to the Center for Disease Control and Prevention (CDC), each year millions of illnesses throughout the world can be traced to foodborne pathogens. Recently, in the USA, the Foodborne Diseases Active Surveillance Network (FoodNet) conducts surveillance at 10 U.S. sites for all infections caused by selected pathogens transmitted commonly through food. A total of 19,531 infections, 4563 hospitalizations, and 68 deaths associated with foodborne diseases were reported in 2012 [4]. The foods most commonly incriminated include meat and dairy products. Salmonella Typhimurium, Escherichia coli O157:H7 and Shigella spp. are among the major foodborne pathogens affecting people worldwide [5].

All foodborne pathogens are important. However, they can be more or less hazardous based on different situations. For example, some foodborne pathogens cause serious illness with a low infectious dose, such as Salmonella spp. and E. coli O157:H7 [6]. Interestingly, Gram-negative bacterial pathogens account for approximately 69% of the cases of bacterial food-borne disease [7]. Except for listeriosis, almost all food-borne bacterial infections can be attributed to the ingestion of viable, gram-negative, enteric pathogens [7].

Foodborne pathogens also lead to an economic burden every year. According to Scharff [8] in the USA 77.7 billion dollars was lost annually to investigate foodborne illnesses associated with 31 foodborne pathogens and spoilage microorganisms which lead to a serious economic loss.

Although foodborne illnesses are caused by a wide range of foods, meat and dairy products industry remains the biggest safety concern and a focal point in many aspects. So, the objective of current study was conducted to evaluate the prevalence of the Gram-negative bacteria from these products at the end of their shelf-life. To give highlighted data throw more light where food regulatory jurisdictions should focus future educational activities. As well as, to assess food safety and maintenance public health by avoidance of the risk associated with such foods and can reduce economic losses by the early detection of inadequate food products.

2. Material and Methods

2.1. Samples Collection

During November 2016 and April 2017, a total of 300 food samples (180 meat products samples and 120 dairy products samples) were randomly collected from 40 different markets and specialty food shops from Cairo and Giza governorates in Egypt. The meat products samples were included: (60 Beef burger, 60 sausage, and 60 Luncheon samples). The dairy products samples were included: (60 Turkey cheese and 60 White cheese samples). All samples were collected in sterile plastic bags and transferred in ice boxes, and the samples were named, indexed and then investigated microbiologically.

2.2. Samples Preparation

Twenty-five grams of each sample were mixed and homogenized in the stomacher and diluted with (225 ml) buffered peptone water or sterile saline solution to make the sufficient dilutions for the microbiological analysis. Ten-fold dilutions of homogenates samples were prepared and inoculated onto appropriate media [9].

2.3. Isolation of Microorganisms from Meat and Dairy Samples

2.3.1. Detection and Enumeration of Enterobacteriaceae

The prevalence of Enterobacteriaceae was determined by spreading 0.1 ml of each sufficient dilution (dilution usually 10^-1-10^-4) on the surface of Violet Red Bile Glucose Agar (Oxoid; CM 485) and incubated at 37°C for 24 hr round purple colonies, surrounded by a purple halo, were considered to be Enterobacteriaceae [9].

2.3.2. Detection and Enumeration of Coliform Group

The coliform group was determined by spreading 0.1 ml of each sufficient dilution using a solid medium method onto plates of violet red bile agar media. The inoculated plates were incubated for 24 hr at 35°C. Coliform group to be counted will produce purple colonies surrounded by purple halos [9].

2.3.3. Isolation and Enumeration of E. coli

Carried out by streaking 0.1 ml of each of sufficient dilution of each food sample onto plates of Eosin Methylene Blue (EMB) (Oxoid; CM0069) and incubated at 37°C for 24 hr. Typical colonies (greenish metallic with dark purple center) were picked up and transferred to nutrient agar slants and incubated at 37°C for 24 hr for further identification [9].

2.3.4. Isolation of Salmonella

Aseptically 25 g of each sample was mixed with 225 ml of sterile buffer peptone water and incubated at 35°C for 24 hr. One to ten ml mixture was transferred to selenite cysteine broth and incubated at 35°C for 72 hr.

Selective platting: Salmonella and Shigella (SS) agar (Oxoid; CM0099) and xylose lysine deoxycholate agar (XLD), (Oxoid; CM0469) plates were inoculated with enriched cultures then incubated at 37°C for 24 hr. Suspected colonies were creamy with or without black centers on SS agar and red with or without black centers on (XLD) agar [9].

2.3.5. Isolation and Enumeration of Pseudomonas spp

Twenty-five g of the sample was homogenized in 225 ml peptone water, and then serial decimal dilutions were prepared. Amount of 0.1 ml of each dilution was spread on Pseudomonas cetrimide, nalidixic acid (CN) agar; Pseudomonas agar base contains 10 ml/l glycerol and selectivity made by inclusion of cetyl trimethyl ammonium bromide (cetrimide; 200 mg/l) and nalidixic acid sodium salt 15 mg/l and were incubated at 25°C for 48 hr. All colonies that developed on the medium were counted and confirmed their identity as Pseudomonas by microscopically and biochemical examinations [10].

2.4. Purification and Identification of Bacterial Isolates

Bacterial colonies obtained were purified by a streak-plate method on nutrient agar medium. Pure isolates were maintained on slants of the same medium at 4°C for subsequent identification.

2.4.1. Morphological and Biochemical Identification

Almost all microscopically examinations and biochemical testing used for identification were carried out according to Bergey'smanual [11], Collins and Lyne [12] and Cheesbrough [13].

2.4.2. Identification by Analytical Profile Index (API) Strips

The Analytical Profile Index (API) strips (API 20E) obtained from (bioMérieux, France), were used as a biochemical system for identification of Enterobacteriaceae and other Gram-negative rod bacteria. The API strip consists of micro-tubes containing dehydrated substrates. These tests are inoculated with a bacterial suspension that reconstitutes the media. The strips were incubated for 18-24 hr at 37°C. During incubation; metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions were read according to the reading table, and the identification was obtained by referring to the Analytical Profile Index [14].

2.5. Antibiotic Sensitivity Test for Bacterial Isolates

In this investigation, 18 commercially prepared antibiotic discs (6 mm in diameter) belonging to different groups were chosen for investigating their potency against bacterial isolates. The discs were obtained from (Oxoid, UK.) properties of the target antibiotics are listed in the table (1). In this test, the standard Kirby-Bauer disk diffusion method according to Bauer et al. [15] was performed in which, four to five similar colonies from overnight growth plates were transferred aseptically in sterile distilled water and vigorously agitated to give a turbidity that matches the 0.5 McFarland standard (approximately 10⁸ cfu/ml) according to D’Amato and Hochstein [16].

Table 1. Antibiotics for sensitivity test

Within 15min, sterile cotton swab dipped into the culture suspension was used for inoculating the surface of solidified Mueller-Hinton agar plates [17]. Antibiotic discs were dispensed onto the inoculated plate surface and gently pressed down using a sterile forceps to ensure complete contact with the agar. Within 15 minutes of applying discs, the inverted plates were aerobically incubated at 37°C for 24 hr. The resulted diameters of inhibition zones around the antibiotic discs were measured to nearest whole mm and interpreted according to protocols standardized for the assay of antibiotic compounds as guided by the National Committee for Clinical Laboratory Standards “NCCLS.” The results were categorized as R (resistant), I (intermediate sensitive), and S (sensitive) [17, 18].

3. Results and Discussion

3.1. Incidence of Gram-Negative Bacteria in Meat Products

The obtained results of the incidence of Gram-negative foodborne bacteria were summarized in table (2) and illustrated in figure (1). The mean value of Enterobacteriaceae; count on a beef burger, sausage and luncheon samples were 3.21, 3.77 and 4.55 cfug⁻¹, respectively, with incidence percentages of positive samples were 30, 38.3 and 56.6%, respectively. The average count of the total Coliform group; on a beef burger, oriental sausage and luncheon samples were 3.36, 4.61 and 4.89 cfug⁻¹, respectively, contained with contamination percentages 20, 28.3 and 35%, respectively (Table 2 & Figure 1). These findings corroborate previous works [19] found the hamburgers were categorized as unsuitable for human consumption in 31.4% of samples, with those testing positive for Enterobacteriaceae and coliform. The presence of Enterobacteriaceae coupled with unhygienic surroundings like sewage, improper waste disposal system, might be the possible sources of food contamination. Members of the family Enterobacteriaceae have been considered a potent cause of foodborne outbreaks, therefore; the presence of members of Enterobacteriaceae in the foods under study might pose a health risk to children and individuals with underlying conditions [20].

Table 2. Incidence of Gram negative bacteria in different meat products

Figure 1. Histogram of the prevalence of isolated Gram-negative foodborne bacteria from meat products

E. coli count; the average count of E. coli was 3.92, 3.69 and 3.73 cfug⁻¹ with incidence contamination percentages of positive samples 20, 28 and 49% of beef burger, sausage and luncheon samples, respectively (Table 2 & Figure 1). The prevalence of E. coli could be attributed to the use of contaminated water during the different stages of processing, in this respect the initial contaminated water used for washing the raw meat is also used for washing hands and utensils used in production, water is a major means by which E. coli are spread [21].

Salmonella typhi was found in low-level contamination of meat products. Where, contamination percentages ranged from 0 to 14%, according to the type of meat product, the number of positive samples was 7 and 14 samples with incidence percentages 11.6% and 23.3% of sausage and luncheon samples, respectively, while the microorganism not detected in beef burger (Table 2 & Figure 1). Unwashed hands of an infected food handler may also contaminate food, and improper preparation and handling of foods at food service establishments are primary factors for Salmonella outbreaks [22]. These results are in harmony with Zhao et al. [23], they isolated in 19–54% of cattle carcasses, 1.9% of beef samples at retail and 4.2% of retail chicken samples. During weeks 1-52, 2002, there were 49 cases as of January 16, 2003, of salmonellosis in Trinidad and Tobago [24]. Lengeler et al. [25] reported the presence of Gram-negative facultative anaerobes including Klebsiella (K. penumoniae), Salmonella (S. typhi) and Proteus (P. vulgaris) in investigated food samples.

Pseudomonas aeruginosa was detected in only one sample of beef burger, while oriental sausage and luncheon samples showed contamination with incidence percentages were 8.33% and 18.33%, respectively. The mean count of the microorganism in the different samples was 3.42, 3.88 and 3.64 cfug⁻¹. The luncheon samples were exhibited the highest contamination percentage with E. coli, S. typhi and P. aeruginosa (Table 2 & Figure 1). Our findings are in an appositive relationship with the previous results obtained by Samson et al. [26], they reported, the presence of Gram-negative aerobes including Campylobacter (C. jejuni and C. coli), Pseudomonas (P. aeruginosa, P. fluorescens, and P. putida) in different meat samples examined.

3.2. Incidence of Gram-Negative Bacteria in Dairy Products

The results presented in table (3) and illustrated in figure (2) showed the incidence of Gram-negative foodborne bacteria in dairy samples. These results exhibited that, the average count of Enterobacteriaceae; were 4.46 and 3.82 cfug⁻¹ with incidence percentages of positive samples 65 and 31.6% of turkey cheese and white cheese samples, respectively.

Table 3. Incidence of some Gram-negative bacteria in dairy products

Figure 2. Histogram of the prevalence of isolated Gram-negative bacteria from dairy products

Coliform group count; the results showed that the average count was 4.24 and 3.25 cfug⁻¹ for turkey cheese and white cheese samples, respectively. The highest contamination percentage was observed in turkey cheese (70%), while the incidence contamination percentage of white cheese samples was 30% (Table 3 & Figure 2). The incidence of Enterobacteriaceae and Coliform group is useful indicators of hygiene of post processing contamination of processed foods as those bacteria coming from equipment's or contact with raw foods. Similar results were observed by Hassan et al. [27], they showed that the mean value of Enterobacteriaceae count ranged from 3.9x10² to 1x10³ for yogurt and Feta cheese. Also, the contamination percentage of the coliform group, are in consistent with Mohammed et al. [28], where showed that, the mean of coliform log no., was 6.28 cfug⁻¹, while the contamination percentage with coliform was 73.33% of investigated samples.

E. coli count; the mean count contained in turkey cheese and white cheese samples were 3.99 and 3.77 cfug⁻¹ with incidence contamination percentages 80 and 65%, respectively (Table 3 & Figure 2). These findings corroborate with previous other studies in Egypt, where the pathogenic E. coli O157:H7 has been isolated from 19% of the total white cheese samples [29].

S. typhi; showed contamination percentage 11.6% of turkey cheese samples and 3.3% of white cheese samples (Table 3 & Figure 2). The presence of Salmonella spp. in dairy products may be due to the using of raw milk for production accompanied by improper sanitary practices during manufacturing, handling and selling. The isolation of this pathogen hence these foods could be of high risk in transmitting enteric pathogens. These results are supported by the findings of De Buyser et al. [30], they found that Salmonella spp., were responsible for 29 outbreaks.

P. aeruginosa; was detected in 21 samples of turkey cheese, with incidence contamination percentage 35% and mean count 3.98 cfug⁻¹. While in white cheese, the number of contaminated samples were 13 samples with incidence percentage 21.6% and mean count 4.31 cfug⁻¹ (Table 3 & Figure 2). These results are in consistence with the previous study by Leriche et al. [31], they found that, thirty Pseudomonas spp. strains isolated from milk, water, cheese center and cheese surface in two traditional workshops manufacturing raw milk.

3.3. Identification of Bacterial Isolates

The bacterial isolates from meat and dairy food samples were taken to be identified according to their morphological, cultural characteristics and consumption of broth manual some biochemical tests according to Bergey's manual [11]. The results obtained from biochemical identification indicated that there are three different groups of foodborne bacterial isolates. The results of morphological and biochemical characteristics of bacterial isolates were given in table (4). The identified bacterial isolates from all collected food samples (meat & dairy) products belonged to two main bacterial families Enterobacteriaceae and Pseudomonadaceae. From each group, we take one isolate for confirming the identification of isolated pathogenic bacteria by using the Analytical Profile Index (API) system (Table 5).

Table 4. Morphological and biochemical characteristics of bacterial isolates

Table 5. API identification of E. coli, S. typhi and P. aeruginosa

3.4. Antibiotic Sensitivity of Foodborne Bacterial Isolates

The antibiotic sensitivity of tested bacterial isolates showed different susceptibilities ranging from sensitive, intermediate and resistant against different tested antibiotics as indicated in the table (6).

Table 6. Antibiotic sensitivity profiles of foodborne bacterial isolates

3.5. Prevalence of Pathogenic Foodborne Bacteria in Various Food Types

The results of morphological and biochemical characteristics of bacterial isolates from all collected food samples (meat & dairy) products belonged to two main bacterial families (Enterobacteriaceae and Pseudomonadaceae) depicts the occurrence of possible pathogens in the 300 food samples tested. Bacterial growth was observed in all the food types; the most prevalent bacteria were E. coli (184) isolates, P. aeruginosa were 51 isolates, while S. typhi exhibited the lowest dominant with (30) isolates (Figure 3).

Figure 3. Total number of isolated bacteria from meat and dairy products

3.6. Distribution of Pathogenic Foodborne Bacteria in Various Food Types

The results summarized in table (7) and illustrated in figure (4) showed the proportional distribution of foodborne bacterial isolates associated with meat and dairy samples examined. Since, E. coli was (184) with (20), (28), (49), (48) and (39) isolates in a beef burger, sausage, luncheon, turkey cheese and in white cheese samples, respectively.

As well as, P. aeruginosa was (51) isolates with (1), (5), (11), (21) and (13) isolates in a beef burger, sausage, luncheon, turkey cheese and white cheese samples, respectively. While, S. typhi showed the lowest dominant percentage (30 isolates) with (7), (14), (7) and (2) isolates in oriental sausage, luncheon and turkey cheese and white cheese samples, respectively, while not detected in beef burger samples.

These trends of results are harmony with Stopforth et al. [32], they Analyzed 1,022 fresh beef samples for levels of microbial populations (total aerobic plate count, total coliform count, and E. coli count) and the presence or absence of E. coli and Salmonella. The mean incidence rates of E. coli and Salmonella on raw beef cuts were 0.3 and 2.2%, respectively. Levels of the total coliform count and E. coli count did not (P > or = 0.05) appear to be associated with the presence of E. coli and Salmonella on fresh beef cuts. While our results were not accordance with Nyenje et al. [33], they assessed the microbiological quality of 252 samples which included rice, pies, beef and chicken stew, the organisms isolated included: Listeria spp. (22%), Enterobacter spp. (18%), Aeromonas hydrophila (12%), Klebsiella oxytoca (8%), Proteus mirabilis (6.3%), Staphylococcus aureus (3.2%) and Pseudomonas luteola (2.4%). Interestingly, Salmonella spp. and Escherichia coli were not isolated in any of the samples, the total number of bacterial isolates were 588 isolates.

Table 7. Bacteria distribution in the various food samples examined

Figure 4. Histogram of positive samples with foodborne bacteria of meat and dairy products