YERSINIA IN SOME EGYPTIAN READY-TO-EAT MEAT PRODUCTS

YERSINIA IN SOME EGYPTIAN READY-TO-EAT MEAT PRODUCTS

SH.M.S. ABD-ALLAH

Department of Food Hygiene (Meat Hygiene), Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt

E.mail: sherief74@yahoo.com

INTRODUCTION

The study of the microbial quality of ready-to-eat meat products including cured meat products is important from the public health point of view as most of these food products consumed without any need for further cooking. To ensure the safety of these foods, periodic surveillance to determine pathogenic agents is necessary (Bryan et al., 1968).

The genus Yersinia is a typical member of the Enterobacteriaceae although it has a number of distinctive features. Yersinias are low temperature pathogens and are able to grow at 4ºC, therefore in cold chain food products they could offer a potential food safety hazard (Varnam and Evans, 1991; Grahek-Ogden et al., 2007). Yersinia are widely distributed throughout the environment and have been isolated from raw milk, sewage-contaminated water, soil, sea-food, humans, and many worm-blooded animals such as poultry and especially pigs (Schmidt and Rodrick, 2003).

Of the many species that comprise the genus Yersinia, three are important from the human pathogenicity point of view, namely Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Yersinia pseudotuberculosis, and Y. enterocolitica are foodborne pathogens (Boer, 1992; Sprague and Neubauer, 2005; Tennant et al., 2005). Yersinia enterocolitica "an important entero-pathogen" can cause acute enteritis (especially in children), enterocolitis, mesenteric lymphadenitis, and terminal ileitis (Bottone, 1997). Some other species of Yersinia including Y. fredericksenii and Y. kristensenii considered to be environmental organisms (nonpathogen) have not been studied extensively due to the absence of classical Yersinia virulence markers. They were occasionally reported to be associated with illness in human and were isolated from some patients as well as Y. enterocolitica considering them as opportunistic (Varnam and Evans, 1991; Sulakvelidze, 2000).

The pathogenic bacterium Y. enterocolitica has become increasingly important as a food contaminant. Of special significance in food hygiene is the ability of Y. enterocolitica to grow in refrigerated foods at temperatures close to 0˚C (Kapperud, 1991). Worldwide surveillance data show an extensive increase in the number of non-outbreak-related isolates and cases of yersiniosis reported. This notice has led to the referral of Y. enterocolitica as a potential emerging enteric human pathogen worldwide (Ostroff, 1995; Tauxe, 1997).

In most developing countries, little is known about the incidence of foodborne diseases or pathogens and associated foods. Therefore, the determination of prevalence of different foodborne pathogens in foods will indicate the potential health risks to consumers (CDC, 1983).

In Egypt, basterma and luncheon, ready-to-eat meat products, are kept in the refrigerators during their storage in the markets especially after the packages is being opened. As yersiniae are widely distributed in the environment, they can contaminate such meat products especially with poor food handling procedures, and they are able to proliferate at 4ºC, which may constitute a public health hazard. Therefore the present study was conducted to investigate Yersinia in those meat products.

MATERIALS and METHODS

1. Sample collection:

A total of 70 basterma and 30 luncheon samples were randomly collected from different localities (mini-markets, supermarkets) in Assiut city, Assiut, Egypt. The samples were dispatched directly to the laboratory where they were investigated with a minimum of delay.

1. Sample preparation:

At laboratory each sample was divided into two parts. One part used for the sensory and the chemical assessments and the other part used for the bacteriological (Yersinia) investigation.

1. Sensory and chemical assessments:

Organoleptic assessment of the samples was done using 9-point hedonic scale according to Meilgaard et al. (2007). The samples pH were detected according to Lyhs et al. (1998) using EC-pH Scan 2 (EUTECH INSTRUMENTS 59000-20) at room temperature. Sodium chloride contents of the samples were determined according the method described by AOAC (1995) using one gram of the thoroughly mixed sample.

4. Yersinia isolation:

4.1. Cold and selective enrichment:

The sample was thoroughly mixed in a sterile mortar. Ten grams of the sample were weighed under sterile conditions and inoculated into 90ml of sterile trypticase soya broth (TSB) supplemented with yersinia selective supplement (Oxoid, SR0106E); mixed well and incubated at 4ºC for up to 14 days for cold and selective enrichment of Yersinia (Greenwood and Hooper, 1989).

4.2. Selective plating:

Isolation of Yersinia was done by streaking a loopful from the previous enriched broth onto the surface of selective agar media (Fredriksson-Ahomaa and Korkeala, 2003). Yersinia isolation agar (HIMEDIA, M564) was used for plating and the inoculated plates were incubated at 30ºC for 24 – 48 hours. Plates showing colonies resembling Yersinia (well established colonial growth and/or red colonies surrounded by a transparent border "bull eye like") were considered suspected positive for Yersinia. The negative plates show no or weak bacterial growth (non-characteristic).

4.3. Strains purification:

Colonies resembling Yersinia on the selective agar plates (at least 2 colonies from each plate) were sub-cultured onto nutrient agar plates, incubated at 28ºC for 24 hours for purification. Separate colonies were picked up from the previous plates and sub-cultured onto nutrient agar slants, incubated at 28ºC for 24 hours, then kept in the refrigerator for further identification.

5. Confirmation of colonies from selective agar plates:

To confirm the obtained isolates as Yersinia, all the pure isolates on the agar slants were sub-cultured for refreshment and examined for gram staining, urease activity, Kligler's Iron agar reaction, and utilization of glucose (gas production) (Krieg and Holt, 1984).

6. Biochemical characterization of Yersinia strains:

All the isolates which were confirmed as the genus Yersinia were submitted to further biochemical testing to identify its species. Activities of lysine decarboxylase (Møller), ornithine decarboxylase (Møller), urease, and utilization of rhamnose, sucrose, cellobiose, sorbitol, and raffinose were evaluated. Further analyses were also conducted applying esculin hydrolysis, indole and Vogus-Proskauer tests (25°C) (Krieg and Holt, 1984).

7. Biotyping of Yersinia enterocolitica:

Yersinia enterocolitica isolates were biotyped according to lipase activity (hydrolysis of Tween 80), Nitrate reduction, indole production, and utilization of D-xylose and D-trehalose (acid production) (Varnam and Evans, 1991).

RESULTS

Sensory evaluation of the examined basterma and luncheon samples revealed that all the examined samples were organoleptically accepted (data not showen).

The results recorded in Table 1 revealed that, the pH values of the examined basterma samples ranged from 3.3 to 6.1 with a mean value of 4.69±0.12. Luncheon samples had pH values ranged from 4.5 to 6.9 with a mean value of 5.85±0.1. Concerning the sodium chloride contents of the samples, they ranged from 4.07 to 12.34 and from 0.65 to 2.93 with a mean value of 7.23±0.21 and 1.7±0.09 for basterma and luncheon samples, respectively (Table 1).

The data presented in Table 2 declared that, out of the 100 examined samples, 39 were suspected positive for Yersinia (out of them only 19 were confirmed positive), and 61 were negative.  Out of the 70 examined basterma samples 28 (40%) were suspected positive and 42 (60%) were negative. However, only 13 (18.57%) out of the 70 basterma samples were confirmed positive for Yersinia. On the other hand, 11 (36.67%) out of the 30 examined luncheon samples were suspected positive and 19 (63.33%) were negative. Only 6 (20%) of the examined luncheon samples were confirmed positive for Yersinia.

A total of 98 suspected Yersinia isolates were obtained from the suspected samples (74 isolates from basterma samples and 24 from luncheon samples). Out of them, only 27 (27.55%) isolates were confirmed morphologically and biochemically as Yersinia and the other 71 (72.45%) isolates were identified biochemically as non-Yersinia strains. Of the 27 isolates confirmed as Yersinia, 19 were obtained from basterma samples and 8 from luncheon samples (Table 2).

The 27 isolates of Yersinia were further characterized into Y. enterocolitica (10 strains), Y. frederiksenii (5 strains), Y. intermedia (2 strains), and Y. kristensenii (10 strains). Of the 19 strains from basterma 6, 4, 2, and 7 strains were identified as Y. enterocolitica, Y. frederiksenii, Y. intermedia, and Y. kristensenii, respectively. The 8 luncheon isolates were identified as Y. enterocolitica (4 strains), Y. frederiksenii (1 strain), and Y. kristensenii (3 strains), as shown in Table 3. 

Yersinia enterocolitica showed the highest incidence from the investigated samples. It was found in 6 (8.57%) and 4 (13.33%) of the examined basterma and luncheon samples, respectively. However, Y. frederiksenii was detected in 3 (4.29%) basterma and 1 (3.33%) luncheon samples. Yersinia intermedia was detected in 2 (2.86%) of the basterma samples but not in any of the luncheon samples. On the other hand, Y. kristensenii was isolated from 5 (7.14%) basterma and 2 (6.67%) luncheon samples. In a total, Y. enterocolitica was detected in 10% of the all examined samples, while Y. frederiksenii, Y. intermedia, and Y. kristensenii were detected in 4, 2, and 7% of the examined samples, respectively (Table 4).

The 10 identified Yersinia enterocolitica strains from the examined samples were further biotyped into 6, 1, and 3 strains of biovars 1, 3 and 4, respectively. The 6 strains from basterma were biotyped into biovar1 (3 strains, 50%), biovar 3 (1 strain, 16.67%) and biovar 4 (2 strains, 33.33%). Likewise, the 4 Y. enterocolitica strains from luncheon samples were biotyped into 3 (75%) strains of biovar 1and 1 (25%) strain of biovar 4, as declared in Table 5.

As shown in Table 6, the incidence rates of Y. enterocolitica biovars from the examined samples were 6, 1, and 3% for the biovars 1, 3, and 4, respectively. From basterma samples the rates were 4.29% for the biovar 1, 1.43% for the biovar 3, and 2.86% for the biovars 4. While, form luncheon samples, the rates were 10 and 3.33% for the biovars 1 and 4, respectively. The biovar 3 could not be identified from any of the examined luncheon samples.

Table1: Statistical traits for the pH values and the sodium chloride contents of the examined basterma and luncheon samples

* Number of samples

SE= standard error

Table 2: Occurrence of Yersinia in the examined samples

Table 3: Biochemically characterized Yersinia isolates from the examined samples

Table 4: Incidence of the identified Yersinia strains from the examined samples

Table 5: Yersinia enterocolitica biovars

Table 6: Incidence of the Yersinia enterocolitica biovars from the examined samples

DISCUSSION

Ready to eat meat products consumed without any further cooking may constitute a potential health hazards transmitting many pathogens to humans.

The pH of the food and its content of sodium chloride are among the factors that limit the bacterial growth and determine which species that can grow in it. Yersinia can grow at pH range from 4–9 and at sodium chloride concentrations up to 7% (Varnam and Evans, 1991).

The pH mean value for basterma samples "4.69", in the current study, was lower than those detected by Aiedia (1995) "5.40", El Khateib (1997) "5.70", and Abd El-Gafar (1999) "5.32". Concerning their sodium chloride content mean value "7.23%", nearly similar results were detected by Mousa et al. (1993), Aiedia (1995) and Abd El-Gafar (1999). However, higher mean values "8.3% and 8.1%" were reported by El Khateib (1997) and Hassan (1997), respectively. As for luncheon samples, higher pH mean value (6.2) and lower sodium chloride content mean value (1.2) were detected by Abd El-Aziz (2004) in 50 samples of luncheon collected at Assiut city, Egypt.

The pH values (3.3 – 6.1) and sodium chloride contents (4.07 – 12.34) of basterma samples seemed to be less suitable for the growth and multiplication of Yersinia. However, the pH "4.5 - 6.9" and sodium chloride contents "0.65 – 2.93" of luncheon samples seemed to be more suitable. This may explain the lower incidence of Yersinia from basterma (18.57%) than from luncheon (20%) samples. Likewise, it may explain the low percentage of Yersinia isolates from basterma (25.68%) than from luncheon (33.33%) samples. The presence of garlic in the coat layer of basterma may also play a role.

Food-borne pathogenic Yersinia is facultative anaerobic, gram-negative Enterobacteriaceae and is isolated frequently from soil, water,animals, and foods (Ackers et al., 2000; Sharma et al., 2003). The consumption of food contaminated with Yersinia can lead to gastrointestinal infections. Cold chain food productscould offer a potential food safety hazard as Yersinia is able to grow at 4ºC "psychrophilic organism" (Aulisio et al., 1983; Grahek-Ogden et al., 2007).

The total incidence of Yersinia from the examined samples in the current study was 19%. This is at variance with findings recorded by Tassinari et al. (1994) "40%", Abd El-Malek (2001) "11.4%", and Mohamed et al. (2012) "10%" in a variety of meat and meat products samples. Lower incidence of Yersinia was recorded by Mohamed et al. (2012) in basterma (3.3%), and by the same authors and by Abd El-Malek (2001) (6.7% and 8.6%, respectively) in luncheon samples collected at Assiut city.

The variation of incidences between the investigators may be due to the differences in the geographical distribution of Yersinia spp., methods of isolation and diversity of kind of samples. The true incidence of yersiniosis is uncertain as few outbreaks of food-borne illness are investigated and a long incubation period may be required using cold enrichment to recover certain strains from food (ICMSF, 1996).

Yersinia enterocolitica is thought to be a significant food-borne pathogen although the incidence of the pathogenic isolates in food is low (De Boer, 1992; Fredriksson-Ahomaa et al., 2001). Its ability to multiply at refrigeration temperatures makes it of significant importance in food hygiene (Lee et al., 1981). In the current study, Y. enterocolitica was detected at an incidence rate of 10% in the examined samples, which disagree with Hudson et al. (1992), who found 3.4% of 203 samples of ready-to-eat fleshfoods purchased from retail outlets at Hamilton, New Zealand, positive for Y. enterocolitica. The authors assumed that in the case of cooked fleshfoods, most contamination occurs post-cooking and contamination rates are increased by poor food handling procedures.

In basterma samples the incidence rate of Y. enterocolitica was 8.57%. Lower incidences were recorded by Abou El-Ela (1994) and Mohamed et al. (2012). For luncheon samples, the obtained rate was 13.33%, which is higher than those reported by El-Gohary et al. (1993), Abou El-Ela (1994), Ahmed and Mohamed (1998), Abd El-Malek (2001), and Mohamed et al. (2012) who reported rates of 10%, 6.6%, 7.5%, 2.9%, and 6.7%, respectively.

The variation between the present findings and those obtained by other investigators may be attributed to several factors including differences in isolation and identification methods and diversity of hygienic condition and competing microflora of samples (Güven et al., 2010).

Yersinia enterocolitica is a common food-borne enteric pathogen found in water, dairy products, and meats (Tauxe et al., 1987; Ackers et al., 2000). It causes human infections whose symptoms include diarrhea, terminal ileitis, mesenteric lymphadenitis, arthritis, and septicemia (Krieg and Holt, 1984).

Biotyping of Y. enterocolitica is of considerable importance as a means of discriminating within the species (Varnam and Evans, 1991). Yersinia enterocolitica, is highly heterogeneous species and can be divided into several bioserotypes, only a few of which are known to associate with human disease (Bottone, 1997; Robins-Browne, 1997). Most Y. enterocolitica strains associated with human yersiniosis belong to bioserotypes 1B/O:8, 2/O:5,27, 2/O:9, 3/O:3, 2/O:5,27 and 4/O:3 with bioserotype 4/O3 dominated globally (Brubaker, 1991; Bottone, 1999). Environmental biovar 1 of Yersinia enterocolitica were isolated from water, soil, food, faeces, humans and animals, and they are nonpathogenic (Varnam and Evans, 1991). 

In the current study, biovars 1, 3, 4 were detected from basterma samples, while biovars 1, 4 were obtained from luncheon samples at varying rates. Biovar 1 was the highest in number (6 strains) and incidence rate (6%), while biovar 3 was the lowest (1 strain; 1%). Mohamed et al. (2012) detected biovar 3 in 11.1 and 2.9% of examined basterma and luncheon samples, respectively, which is higher than the obtained results. Abd El-Malek (2001) isolated biotype 3 at an incidence rate of 22.2% from examined luncheon samples. In the present investigation biovar 3 could not be detected in any of the examined luncheon samples. Abou El-Ela (1994) identified Y. enterocolitica biovar 4 recovered from luncheon samples at percentage of 6.6% that seemed higher than the present findings (3.33%).

Yersinia frederiksenii, Y. intermedia and Y. kristensenii are considered to be environmental organisms. However, they described as opportunistic occasionally associated with illness in humans. Yersinia frederiksenii was isolated from some patients as well as Y. enterocolitica (Varnam and Evans, 1991).

Yersinia intermedia and Y. kristensenii failed to be detected in any of examined basterma and luncheon samples collected at Assiut city by Mohammed et al. (2012), which is partially at variance with the current findings. Abd El-Malek (2001) could not detect Y. intermedia in any of the examined luncheon samples collected at Assiut city, which is in agreement with the obtained results; however he could detect Yersinia kristensenii in luncheon samples at an incidence rate of 5.7% that is seemed slightly lower than the present findings (6.67%). El-Gohary et al. (1993) detected Y. intermedia at a rate of 4% in 50 samples of luncheon collected from Alexandria Governorate.

Yersinia frederiksenii, and Y. intermedia has been isolated mainly from fresh water sources, fish, foods, and occasionally from sick and healthy humans. Strains of Y. kristensenii have been isolated mainly from soil, from various environmental sources (fresh water, foods) and rarely from healthy or sick man and animals (Krieg and Holt, 1983).

On conclusion, the results of this study emphasized that basterma and luncheon available in the markets were contaminated with Yersinia, which may reflect the lack of hygienic supervision. For that, these products may constitute a potential health hazards to human causing sporadic non-outbreak illness of yersiniosis. Good sanitary precautions, hygiene and proper food handling may be necessary to prevent contamination of such products with Yersinia, especially pathogenic ones, and to control the disease. As well, proper heat treatment before consumption "if possible" may be helpful to control the disease.

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