MICROBIOLOGICAL STUDIES ON SALMONELLA MICROORGANISM IN SOME MEAT PRODUCTS

Dept. of Bacteriology,

Animal Health Research Institute, Shebin El-Koom.

Microbiological studies on Salmonella microorganism in some meat products

(With 4 Tables and One Photo)

By

Eman M. Sharaf; Ebtesam M. Mezyed*

and Gehan S. Afifi**

Serology Unit, Animal Health Research Institute, Dokki, Cairo.

**Fac. Vet. Med., Moshotohor, BanhaUniv.

(Received at 24/11/2010)

دراسات ميکروبيولوجية علي ميکروب السالمونيلا في بعض منتجات اللحوم

إيمان محمود شرف ، ابتسام محمد فريد ، جيهان سيد عفيفي

تم دراسة مدي تواجد ميکروب السالمونيلا في عينات اللانشون والسجق واللحم المفروم المجمد في 50 عينة من کل نوع وتم عزل السالمونيلا بنسبة صفر و8% و 6% من اللانشون والسجق واللحم المفروم المجمد علي التوالي وتم تصنيف السالمونيلا إلي سالمونيلا تيفيوريم وانتريديدس وتم إجراء اختبار الحساسية لکل منها ووجد ان کل منهما حساس الى الاموکسيللين والسيفروکسيم صوديوم والسيبروفلوکساسين والدانوفلوکساسين والاوکسى تتراسيکلين وکل منهما مقاوم للاستربتومايسين وأيضا تم إجراء اختبار البلمرة المتسلسل وکان إيجابي بالنسبة للنوعين.

Summary

Hundred and fifty random samples of various meat products represented by luncheon, sausage and frozen minced meat (50 of each) were collected from different markets for detection of Salmonella spp., Obtained results declared that Salmonellacould be detected in zero, 8 and 6% of the examined samples of luncheon, sausage and frozen minced meat respectively. The recovered salmonellae were serotyped as Salmonella typhimurium and Salmonella enteritidis. The in vitro senstivity tests for theisolated bacteria were determined, Salmonella typhimurium and Salmonella enteritidis were sensitive to Amoxicillin, cefuroxime sod. Ciprofloxacin, Danofloxacin and oxytetracycline and were resistant to streptomycin. Salmonella typhimurium and Salmonella enteritidis can be detected by Real-Time PCR when used Quantitect probe RT-PCR kit cat. no. 20 4443 which based on fluoroqenic primers and probe (TAMRA dye).

Key words: Salmonella microorganism, meat products, minced meat, sausage, luncheon, antibiogram, PCR.

Introduction

Meat products such as luncheon, sausage and minced meat have a popularity because they represent quick, easy prepared meat meals and solve the problem of the storage in fresh meat of high price which is not within the reach of large number of families with limited income (Mohamed 2006). On the other side, meat products are liable to harbour different types of microorganisms through along chain of handling, processing, distribution and storage as well as preparation (Hassanien, 2004). Within this respect meat in general and poultry in particular are the commonest source of food borne diseases and have been frequently linked to outbreaks of food poisoning by Salmonella (Antunes et al., 2003). Salmonella spp. typically cause an intestinal infection with or without fever, the spread to human is usually caused by consumption of contaminated food stuffs (Bhan et al., 2005). Salmonella is one the most common cause of food borne diseases (Tirado and Schmidt, 2001) for this reason, the number of rapid test methods for Salmonella has grown rapidly in the last decade. PCR and real time PCR have become powerful tools for detection of pathogens in food. (Malorny et al., 2003 and 2004). The real time PCR in food borne outbreak investigations provides an opportunity in food and clinical settings (Burkhard et al., 2004). This study was undertaken for isolation ofSalmonellafrom some meat products and identification of the isolates by real- time PCR.

MaterialS and Method

I- Collection of samples:

A total of 150 random samples of luncheon, sausage and frozen raw minced meat( 50 of each) were collected from different markets in Egypt an examined for the presence of Salmonellaspecies.

II- Isolation and Identification. (FAO, 1979).

The method for isolation and identification of Salmonella recommended by Edwards and Ewing (1972), Cowan and Steel (1975) and FAO (1979) was followed.

Pre-enrichment

25gm of each sample were blended with 225ml of buffered peptone water and transfered aseptically to a sterile 500ml flask then incubated at 37°C for 10-20h.

Enrichment

10ml of each pre-enrichment medium were transfered to 100ml tetrathionat broth medium and another 10ml to 100ml selenite F. broth medium previously warmed to 42-43°C and incubated at 42-43°C for 48h.

Plating on selective media:

Brilliant green agar, MacConkey agar and Salmonella-shigella agar media were streaked from each enrichment flask and incubated at 37°C for 24h., then examined for typical colonies for Salmonella.

Typical or suspected colonies were selected from each selective medium and streaked on nutrient agar medium which incubated at 37°C for 24h.

Morphological, biochemical and serological confirmation were performed according to Edwards and Ewing (1972), and Cowan and Steel (1975).

III- Antibiogram:

Salmonella isolates were tested for sensitivity to 15 antibiotics by the disc and agar diffusion method. The interpretation of the results was carried out according to NCCLS (2002).

V- Real-time PCR (Petra et al., 2005):

A real-time PCR assay was developed based on fluorogenic primers and probe, sal-F (5^, -GCGTTCTGAACCTTTGGTAATAA-3^,), sal-R (5^,-CGTTCGGGCAATTGGTTA-3^,),and probe (5^,-FAM-TGGCGGTGGGTTTGTTGTCTTCT-TAMRA-3^,) used for amplification of a 102-pbregio on of the inVA gene of Salmonella. The PCR mixture consisted of 12.5µl 2X Quantitect probe RT-PCR master Mix, 0.2 µl primer F 50 pmol, 0.2V primer R 50 pmol, 0.25V probe 30 pmol 4-5 µl RNase free water 2-5 µl template RNA (Quantitect probe RT-PCR kit catalogue no-20 4443).

Results

Table 1: Incidence of Salmonella in the examined samples of meat products

Table 2: Serotyping of Salmonella isolated from the examined samples of meat products (N= 50)

Table 3: Antigenic formula of Salmonella recovered from meat products samples.

Table 4: Antibiogram patterns of isolated Salmonella serovars

     S= sensitive

    M= Moderate sensitivity                       R= Resistant

Photo 1: Analysis of Real-time PCR for Salmonella typhimurium and Salmonella enteritidis.

Discussion

Meat and meat products are considered as a major vehicle  of  most reported outbreaks of foodborne disease. Epidemiology data have identified improperly handled meat products as important vehicles for infection (ICMSF, 1978), Salmonella remains as one of the major food borne health hazards and meat plays an important role, as a reservoir, in disseminating Salmonella (Mohamed, 2006).

In present study a total of one handred and fifty random samples of meat products (50 samples of luncheon, 50 samples of fresh sausage  and 50 samples of frozen minced meat) were examined for salmonellae. Results obtained in Table (1) revealed  zero, 8 and 6%that Salmonella were detected in the examined luncheon, sausage and frozen minced meat respectively. These results agree with that reported by Saleh (1991); Edris (1993); Moussa et al. (1993); Fathi et al. (1994); Aiedia (1995); Abd-El-Aziz et al. (1996); Ouf (2001) and Eleiwa (2003). The result disagrees with that reported by Mohamed (1988) who recorded that salmonellae could not be detected in the examined luncheon samples. From the results recorded in Table (1) it's clear that fresh sausage had higher incidence (8%) of Salmonella contamination followed by frozen minced meat(6%). High incidence of salmonellaein fresh sausage may be due to faults in certain practices of slaughtering and handling processes such as the use of contaminated knives, tools, rags, saws, boards ….etc as well as unhygienic slaughtering, dressing, washing, transporting, handling and cutting in abattoirs and butcher shops. The high incidence of salmonellaein frozen minced meat may be due to cutting and contamination of meat besides the increase in its water and oxygen contents as well as contamination from grinders, dir, packaging materials and hands of workers. Temperature rise (2-4°C) during grinding could also increase the incidence of salmonellae  organisms (Field et al., 1977). The absence of salmonellaein luncheon meat may be due to the addition of food additives such as spices and preservatives, which have an antimicrobial activity and inhibit survival and multiplication of micro-organisms (Libby 1975). This also may be attributed to the exposure to high temperature during processing and cooking procedures.

From the results recorded in Table (2) it's clear that four  Salmonella serovars were identified from sausage samples, two (4%) strains as S. typhimurium and two (4%) strains as S.enteritidis.Nearly similar results were obtained by Rao and Nandy (1977). It's evident that three Salmonella serovars were isolated from the examined frozen minced meat samples and identified as two (4%) strains as S. typhimurium and one (2%) as S. enteritidis. These results agree with that obtained by Gobran(1985).

Antibiogram patterns (Table 4) showed that Salmonella typhimurium isolates were resistant to streptomycin and sensitive to amoxicillin, cefuroxime sod., ciprofloxacin, Danofloxacin, flumequine, Enrofloxacin and oxytetracycline. Salmonella enteritidis was similar to Salmonella typhimurium. These results are similar to those recorded by Frech and Schwarz (1998); Guerra et al. (2000); Wiuff et al. (2000) and Gebreyes et al. (2004).

Photo (2) showed that Salmonella typhimurium and Salmonella enteritidis were detected by Real-time PCR when used fluorogenic primers and TAMARA-probe used for amplification of 102-bp region of the in VA gene of Salmonella (Quantitect probe RT-PCR kit cat.no.20 4443), according to Kura et al. (1999); Livak et al. (1995); Paszko et al. (1997); Hoorfar and Radstrom (2000).Detection probability of Salmonella real- time PCR assay at serially 10- fold- diluted cell concenterations of serotypetyphimurium and enteritidisreference strains was determined in the presence of 150 copies of IAC DNA. Five microliters of each suspension (10⁰ to 10⁶ CFU/ unit) was used as the template in the PCR. The graph shows a sigmoidal fit of data points generated by 30 repetitive PCRs. The real-time PCR in food borne outbreak investigations provides an opportunity for rapid detection of pathogens in food and clinical settings (Burkhard et al., 2004).

Apart from saving time, real-time PCR is sensitive highly specific and offers the potential for quantification, the risk of cross-contamination is significantly reduced and high-through put performance and automation are possible, since no post-PCR manipulations are required (Harnai et al., 1997; Lubeck and Hoorfar, 2003).

From the obtained results we can concluded that the possibility of contamination of meat products with such serious pathogens remains as a public health problem. Thus all precautions of proper sanitation during manufacture, handling and storage of such meat products should be adopted to control these serious pathogens and to obtain a maximum limit of safety to consumers. The RT- PCR may be considered as a rapid, sensitive highly specific and offers the potential for quantification of Salmonella isolates.

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