"Incidence of Listeria Monocytogens in Frozen Beef, Poultery and Fish in Assuit City

Animal Health Research Institute

                   Assuit Regional Laboratory.

"Incidence of Listeria  Monocytogens in Frozen Beef, Poultery and Fish in Assuit City"

"مدي تواجد ميکروب الليستريا مونوسيتوجينز في لحوم الأبقار والدواجن والأسماک المجمدة في مدينة أسيوط"

By

Lubna M. Ebraheem & Manal H. Thabet

لبني محمد أبراهيم ، منال حسن ثابت

الملخص العربي

أجريت هذه الدراسة علي 90 عينة ـ بواقع 30 عينة لکل من اللحوم والدواجن والأسماک المجمدة . حيث جمعت العينات من المحلات المختلفه بمحافظة أسيوط وتم عزل ميکروب الليستريا بنسة 33% في اللحوم المجمدة ، 60% في الدواجن المجمدة ، 53% في الأسماک المجمدة . وتم عد الميکروب حيث وجدت الليستريا بمتوسطات 2.9× 10 ³ ± 1.9 ، 5.65×10 ²± 4.7 ، 12.75×10 ² ± 6.8 / جرام بالترتيب لکل من عينات اللحوم والدواجن والأسماک المجمده علي التوالي تم عزل ميکروب الليستريا مونو سيتوجينز بنسبة 16.6% ، 33% ، 13.3% من کل من العينات السابقه من اللحوم والدواجن والأسماک المجمده بالتريب فکانت أعلي نسبة له في عينات الدواجن . ولقد أوضحت إختبارات الحساسية للمضادات الحيوية أن کل العترات المعزولة کانت مقاومه للجنتاميسن والأمبيسيلين بنسبة 100% ولکنها کانت حساسة للکلورامفينيکول ونورفلوکساين بنسبة 100% ، کما أظهرت العترات نسب مختلفة من الحساسية لکل من ستريبتوميسن ، تيتراسيکلين ، ريفامبين ، سيفادروکسيل ، سيفوتاکسيم .

Summary

            Present study was preformed on 90 frozen samples of meat, poultry and fish (30 of each).  The samples were collected from different shops. in Assiut city . Listeria  species were isolated at different percentages (33%) in frozen meat, (60%) in frozen poultry and (53%) in frozen fish. Also they were counted in the examined samples, the mean counts were 2.9×10³±1.9, 5.65×10² ± 4.7 CFU/g and 12.75×10² ± 6.8 CFU/g of the examined frozen samples of meat, poultry and fish respectively. Listeria  monocytogenes was differentially identified from other Listeria  species and could be isolated at variable percentages (16.6%) in frozen meat, (33%) in frozen poultry and (13.3%) in frozen fish, the study revealed that the incidence of L.monocytogenes was higher in frozen poultry as compared to both frozen meat and fish. The drug susceptibility characterization of L.maonocytogenes cleared that all isolates were resistant (100%) to Gentamicin and Ampicillin but were susceptible (100%) to chloramphenicol and Norfloxacin, while the other used antibiotic show different degree of antimicrobial sensitivity reactions for streptomycin, Tetracycline, Rifampin, Cefadroxil and Cefotaxime.

Introduction

            Listeria  monocytogenes ispresent in soil, water, vegetables, intestinal contents of a variety of birds, fish, insects and other animals. Human Listeriosis is a sporadic disease which is associated with consumption of under-cooked meat, contaminated milk, soft cheese, unwashed raw vegetables and cabbage (Schuchat et al., 1992). Meat and meat products have frequently been contaminated with L. monocytogenes and may serve as vehicle of other pathogenic organisms . In human, the illness may range from mild to severe sickness. The sever forms of human Listeriosis are present as meningoenceophalitis followed by septic infections and occasionally isolated organ involvement. Death is rare in healthy adults but can occur at a rate as high as 30 % in persons at highest risk (Demetrios et al., 1996). L. monocytogenes is the etiologic agent of about 98% of human and 85% of animal cases (Mclauchlin, 1987). Because of its ability to survive and proliferate at refrigeration temperature, L. monocytogenes may cause disease through frozen foods (Schillinger et al., 1991).The organism can grow over the temperature range of about 1 ْc to 45  ْc and the pH range 4.1 to around 9.6, it may be expected to survive in foods for long periods of time (Ryser et al., 1985). The volume of frozen food consumed ( specially frozen meat, poultry and fish ) is growing all the time. Consequently, it was decided to examine these food in order to establish some indication of the incidence of L. monocytogenes and assess wheather the level of contamination might pose any risk to consumers.

Material and Methods

1- Collection of samples :

            A total of 90 samples were collected from different location in Assiut city for examing the presence of Listeria  monocytogenes. The samples comprised 30 of each from frozen meat, poultry and fish. Each sample was warped separately and aseptically in strile polyethylene bag, then labelled and transferred as quickly as possible to the laboratory .

2 – Bacteriological analysis :

A – Isolation : 

            Twenty-fiive grams of each samples were homogenized with 25 of Listeria  enrichement broth in sterile Moulinex type blender equipped with metallic flask for 1 min, and incubated at 37C^о for 48 h. After incubation one loopful was subcultured on Listeria  selective medium (Oxford agar) according to Oxoid Manual (1990).

B – Enumeration :

            Counting of L. monocytogenes was achieved by direct plating of decimal dilutions of prepared samples (APHA, 1992) onto plates of Oxford agar. The plates were incubated at 37C^о for 24-48 h. and typical colonies presumed to be L. monocytogenes  were counted .

C – Identification :

Colonies suspected to be L. monocytogenes were identified according to (koneman et al., 1996) and Quinn et al., (2002) and characterized according to (Margolles et al., 2000) by Gram stain, tumbling motility , V.P., catalase, oxidase, haemolysis on horse blood agar and CAMP test. For further confirmation of L. monocytogenes the isolates were inoculated into 10 % aqueous stock solution of Mannitol, L. Rhamnose and D. xylose as described by Collee and Miles (1989).

3 – Antimicrobial Susceptibility testing :

            All islolates obtained in this study were tested for antimicrobial susceptibility by disc diffusion method as described by Finegold and Martin (1982) using 9 antimicrobial agents, using the following discs, Chloramphenicol (30 µ g), Norfloxacin (10 µ g), Rifampin (5 µ g), Cefadroxil (30 µ g),Cefotaxime (30 µ g) streptomycim (10 µ g), Tetracycline (30 µ g), Gentamicin (10 µ g) and Ampicillin (10 µ g).

Results and Discussion

Table 1: Incidence of Listeria  species and Listeria  monocytogenes in examined samples.

Table 2 : Statisitical values of Listeria  species in examined samples.

Table 3 : Antibiotic sensitivity test for Listeria  monocytogenes isolates

Discussion

Results given in Table 1 revealed that 48.8% of Listeria  spp. were isolated from 44 out of 90 samples of frozen (meat, poultry and fish) 30 of each. The percentage of Listeria  spp. in each product were 33, 60, 53 respectively, while 21% of L.monocytegenes were isolated from 19 out of the same samples, the organism was found in 16.6% of frozen meat, in 33% of frozen poultry and in 13.3% of frozen fish. (Table 1& Fig. 1)

The incidence of Listeria  spp. in frozen meat 33% was lower than that obtained by Elgazzar and sallam (1997) and Hassan et al. 2001 (73.9%). While the percent of L. monocytogenes in the same product was 16.6% which was lower than that recorded by Nicolas and Vidaud (1987) (26.2%) , Hassan et al. 2001 (75%). On the other hand our result was higher than that recorded 0.59% by (Paul et al. 1988) and 6.3% by Scange et al. 2000.

At the same table the incidence of Listeria  spp. in frozen poultry was (60%) which shown to be intermediary between that detected by Virendera et al. 1995 (82%) ; (Mahmood et al. 2003) (17.5%), while the percent of L. monocytogenes in the same product 33% was nearly agreed with that recorded by (Virendra et al. 1995) (46%) . Lower than that obtained by Pini and Gilbert (1988) (60%) and higher than (7.5%) which recorded by (Mahmood et al. 2003).

Table 1 reveals that the percent of Listeria  spp. in frozen fish was 53% which was nearly agreed with that obtained by Weagant et al. 1988 (61%) and was higher than that recorded by Ronda and Thakor 1992 (35%).

Many investigators detected the presence of L. monocytogenes in frozen fish in variable levels as Wong et al. 1990 recorded 10.5% Hala and Hala (2006) recorded 9.3% such results nearly agreed with that obtained in this study 13.3%. While Weagant et al. 1988 recorded 26%.

L. monocytogenes is of greatest concern from the public health point of view. (Dalton et al. 2004) in their studies about food borne disease outbreaks, found that the most frequently implicated vehicles in 173 outbreaks were sea food and L. monocytogenes caused 40% of deaths.

Table 2 shows that the count of Listeria  spp. of the exmined frozen meat samples ranged from 1× 10³ to 4.8 × 10³ with a mean value of 2.9 ×10³ ± 1.9 CFU/g. The level of contamination of the examined frozen poultry samples varied from 1× 10² to 10.3 × 10²  with a mean value of 5.65 × 10² ± 4.7 CFU/g. It is evident from the same table that the count of Listeria  species in frozen fish samples ranged from 6 × 10² to 19.5× 10² with a mean value of 12.75× 10² ± 6.8 CFU/g.

The difference in the obtained results may be due to the differences between strains, the type of freezing employed, the length of time of freezing storage, temperature of freezing also the nature and composition of the food.

The drug susceptibility is one of the important factors of characterization of  L. monocytogenes antibiotic sensitivity testing indicated that chloramphenicol and Norfloxaicn were the most effective antibiotics, while Ampicillin and Gentamycin were not effective antibiotics. Ampicillin and Gentamicin were not effective . The other used antibiotics show different degree of antimicrobial sensitivity reactions (Table 3). Chloramphenicol and Norfloxacin are consider as the antibiotic of choice. Hala and Hala (2006).

In conclusion the information given by the achieved results revealed that incidence of Listeria  spp. and L. monocytogenes was higher in frozen poultry than that in frozen meat and frozen fish(as shown in Fig.1). This may be due to that frozen poultry are considered as anatural reservoir to L. monocytogenes, and also are more liable to be contaminated during their preparation and storage.

The presence of Listeria  in frozen products supports the statement that freezing has no significant effect on the organism. (Sneath et al. 1986).

Reference