Isolation and Identification of Listeria monocytogenes from Retail Broiler Chicken Ready to Eat Meat Products in Sudan

Listeria species are widely distributed in environment and L. monocytogenes are the causal agent of Listeriosis, the disease that can be serious and fatal to human and animals. The objectives of this study were to detect, isolate and identify Listeria monocytogenes from retail broiler chicken ready to eat meat products in restaurants-Khartoum state, Sudan. A total of 250 retail broiler chicken ready to eat meat products were collected from restaurants in Khartoum State, 50 sample from frozen chicken burger, 50 sample from frozen chicken sausages, 50 sample from frozen chicken meat balls (kofta), 50 sample from chicken shawerma and 50 sample from chicken mortedella, Listeria spp. were isolated by the conventional International Organization for Standardization method and L. monocytogenes identified by biochemical test. The results showed that out of total 250 samples, 95 (38%) were found to be contaminated with Listeria spp. the isolation rate was as follows: L. monocytogenes (13.6%), L. ivanovi (20.8%), L. grayi (1.6%), L. seeligeri (0.8%) and L. welshimeri (1.2%). The results presented in this study indicate the contamination of retail broiler chicken ready to eat meat products with L. monocytogenes. This study reported the occurrence and distribution of L. monocytogenes and other Listeria species in retail meat products (frozen chicken burger, frozen chicken sausages, frozen chicken meat balls (kofta), chicken shawerma and chicken mortedella), purchased from restaurants in Khartoum state Sudan.

L. monocytogenes is pathogenic for human, while L. ivanovii is rarely pathogenic for humans (Cummins et al., 1994) L. monocytogenes are the causal agent of Listeriosis, the disease that can be serious and fatal to human.It is a halo tolerant, Gram-positive, facultative anaerobic, non-spore forming rod bacterium (Larson et al., 1999).It can grow in a wide pH range from 4.6 to 9.5 and can grow in low water activity environments as 0.90 (FAO/ WHO, 2004;Buchanan et al., 2004;Gandhi and Chikindas, 2007).Listeria spp.are considered as an important cause of zoonoses infecting many types of animals such as domestic pets, livestock, avian species, rodents, amphibians, fish, and arthropods.The approximate fatality rate is 30% that may increase up to 75% in high risk groups, such as pregnant women, neonates, and immunocompromised adults (Jalali and Abedi, 2007;Mead et al., 1999;Low and Donachie, 1997).Listeriosis is unique disease that represents a considerable public health concern because of its high mortality rate that reaches 20-40% (Wan Norhana et al., 2010a, b;Liu, 2006;McLauchin et al., 2004).Most cases of Listeriosis appear to be foodborne, including those acquired during pregnancy.L. monocytogenes may cross-contaminate RTE meat and poultry products during post-processing steps such as slicing, peeling, and packaging (Murphy et al., 2005).RTE meat and poultry products are widely consumed in Sudan.To our knowledge no published data has been found concerning isolation and characterization of L. monocytogenes recovered from RTE meat and poultry products in Sudan.The nature of strain persistence is unknown but biofilm formation in food-processing facilities could be one of the important reasons (Bresford et al., 2001).High the prevalence of L. monocytogenes in RTE food is commonly reported in high rates in different parts of world.L. monocytogenes was detected in Spanish-style sausage (3.7%), blood sausage (11.1%), cooked meat samples (8.8%), different RTE foods (7.3%) and in in-store-packaged deli meat products (8.5%) (Mena et al., 2004;Vitas and Garcia-Jalon, 2004;Jalali and Abedi, 2007;Garrido et al., 2009).A USDA-FSIS survey published in 2001 showed that 1-10% of retail RTE meat and poultry products were contaminated with L. monocytogenes (Levine et al., 2001).Although RTE are commonly encountered L. monocytogenes (CDC, 1999;Mena et al., 2004;Vitas and Garcia-Jalon, 2004); another study in Australia also reported low prevalence rate where, only 0.2% prevalence of L. monocytogenes was reported in these products (Ross et al., 2009).Published information on the status of food borne listeriosis is very limited in the Sudan.The objectives of this study were to detect, isolate and identify Listeria monocytogenes from retail broiler chicken ready to eat meat products (frozen chicken burger, frozen chicken sausages, frozen chicken meat balls (kofta), chicken shawerma and chicken mortedella) in restaurants-Khartoum state, Sudan.

MATERIALS AND METHODS
Samples: Two hundred fifty retail broiler chicken ready to eat meat products samples were collected from restaurants in Khartoum state were used for detection of L. monocytogenes, fifty samples from frozen chicken burger, 50 samples from frozen chicken sausages, 50 samples from frozen chicken meat balls (kofta), fifty samples from chicken shawerma and 50 sample from chicken mortedella.All Samples were transported to Sudan University of Science Technology College of Veterinary Medicine Microbiology laboratory under aseptic and refrigerated conditions in portable insulated cold-boxes.Samples were kept at 4°C and analyzed within 24 h.

Isolation and identification of L. monocytogenes:
Retail broiler chicken ready to eat meat products samples were tested for the presence of L. monocytogenes following the procedure recommended by using the International Organization for Standardization (ISO, 1996(ISO, , 2004) ) procedure.A 25 g representative portion from each sample was introduced aseptically into a sterile stomacher bag containing 225 mL of Half Fraser Broth (Oxoid, Ltd., Basingstoke, UK, CM0895) (primary enrichment medium) to obtain a 1:10 sample dilution.The samples were then homogenized for 1 min at 260 rpm in a stomacher circulator unit 400 (Seward, UK) followed by incubation for 24 h at 30ºC.After incubation period, 0.1 mL sub-sample from each Half Fraser Broth culture was added to 10 mL of Fraser Broth (Oxoid, CM0895) (secondary enrichment medium), and incubated for 48 h at 37°C.A loopful of the Fraser Broth enrichment culture was streaked on the surface of Chromogenic Listeria Agar (Oxoid, CM1084) and on Listeria Selective Agar (Oxford Formulation) (Oxoid, CM0856).These selective agars were then incubated for up to 48 h at 37ºC.Selective agars were observed for suspected colonies at 24 and 48 h of incubation.Suspected colonies were those that appeared grayish colonies surrounded by black halos and sunken centers with possible greenish sheen on Oxford agar or greenblue colonies surrounded by an opaque halo zone on Chromogenic Listeria agar.Whenever possible, up to 5 suspected colonies showing typical morphology of Listeriae on these isolation media were streaked onto Tryptone Soya Agar (Oxoid, M290) supplemented by 0.6% of Yeast Extract Powder (Oxoid, LP0021) (TSYEA) and incubated at 37ºC for 24 h.The following tests were used for confirmation; Gram's staining, motility test, catalase reaction, and oxidase test.

Confirmation of L. monocytogenes:
Haemolysis test: An inoculating needle was used to stab the Sheep Blood Agar Base (Oxoid, CM0854), supplemented with 7% sterile sheep blood, with a culture taken from a typical colony on TSYEA and incubated at 37°C for 24 h (ISO, 1996).After incubation positive test cultures show narrow, clear and light zones (ß-haemolysis).

Carbohydrate utilization:
The Microbact ™ Listeria 12L Kit System (Oxoid, MB1128A) for rapid biochemical testing.Microbact ™ Listeria 12L Kit System (Oxoid, MB1128A) is a standardized microsubstrate system designed to stimulate conventional biochemical substrates.Each identification strip consists of 12 tests, (11 sugar utilization tests (Esculin, Mannitol, Xylose, Arabitol, Ribose, Rhamonse, Trehalose, Tagatose, Glucose-1-Phosphate, Methyl-D-Glucose, and Methyl-D-Mannose) plus a rapid haemolysis test.The reactions occurring during the incubation period is demonstrated through either a color change in the sugar utilization tests or in the lyses of sheep red blood cells in the haemolysis test.The results were analyzed by Microbact Software (Oxoid, MB1244A) to determine the L. monocytogenes with percent probability number.
A statistically the difference between percentages of contaminated samples of various food groups with examined pathogen microorganisms was calculated.

RESULTS
Isolation of Listeria spp.from retail broiler chicken ready to eat meat products using of conventional method: According to growth on selective media,  1).

DISCUSSION
The real situation of listeriosis in Sudan is unknown, and no information is available on the presence of L. monocytogenes in retail broiler chicken ready to eat meat products in Sudan.
The standard method for isolation and detection of Listeria is the use of enrichment procedure followed by selective media, are in agreement with Vlaemynck et al. (2000) and Beumer and Kusumaningrum (2003).ALOA medium has proved to be a useful and significantly better assay than other media (Oxford agar, UVM agar and PALCAM agar) for the isolation and differentiation of L. monocytogenes from nonpathogenic Listeria species, because L. monocytogenes colonies on ALOA agar exhibited clear halo zone.
The detection of pathogenic L. monocytogenes by this media involves cleavage of the substrate, L-αphosphatidyl-inositol by the virulence factor Phosphatidylinositol-Phospholipase-C (PI-PLC) and Phosphatidylcholin-Phospholipase-C (PC-PLC) produced by pathogenic L. monocytogenes resulting in the formation of a white precipitation zone (halo) around the colony.
Although conventional method of selective culture media are confirmed by the Microbact ™ Listeria 12L identification System (Oxoid, MB1128A) were designed to detect the type of Listeria spp., Bailey et al. (1990) had examined the factors of colonization of broiler chickens with L. monocytogenes (orally inoculated) did not colonize chickens as easily as did Salmonellae or C. jejuni.Younger birds were more susceptible to colonization than older birds, and there was a dose-related colonization response.It is evident that poultry can become contaminated either environmentally during production or farm healthy carrier chickens in the processing plant (Genigegorgis et al., 1989;Bailey et al., 1990).
Processed meats are meat products produced from raw meat and other ingredients, stored under refrigeration or frozen conditions that may need further cooking to stop L. monocytogenes or could be consumed directly without cooking (Tsutomu, 1990).
In Current study, the presence of L. monocytogenes in processed meats could be explained by the inadequate heat treatment to destroy the growth of L. monocytogenes or as a result of post process contamination.Many studies have reported that the prevalence rate in RTE meats ranged from 1.8 to 48% (Wilson, 1995;Bersot et al., 2001;Eleftheriadou et al., 2002;Soultos et al., 2003;Mena et al., 2004;Vitas and Garcia-Jalon, 2004).The growth of L. monocytogenes in meat is highly dependent on the temperature, the pH of the meat, the type of tissue and the type and amount of background micro flora present.Glass and Doyle (1989) found that the growth of L. monocytogenes in meat was highly dependent on product type and pH.
The organism was able to grow well in meat products with a pH value near or above 6.0 while it grew poorly or did not grow in meats that have pH-5.0.L. monocytogenes is able to contaminate RTE meat products by transmitting through post-processing steps including slicing, packaging and freezing.Thus, these foods needs a further heat cooking at homes, hotels, restaurants to eliminate Listeria spp., especially L. monocytogenes.In addition, cross-contamination between raw materials, equipments, utensils, humans, rodents, insects, animals and birds could contribute to the spread of L. monocytogenes in food processing plants (Jemmi and Stephen, 2006).In addition, the ability of L. monocytogenes to form biofilms inside food facility and on the surfaces of food production lines that is quite difficult to be removed and cleaned (Bresford et al., 2001).For example, Listeria spp., especially L. monocytogenes is able to transfer through vacuum and gas-packaged products due to their ability to grow and survive at low temperature (Duffy et al., 1994;Huss et al., 2000).
In present study (16%, 12%, 18%, 4% and 18%) L. monocytogenes isolates were recovered from retail broiler chicken ready to eat meat products in frozen chicken burgar, frozen chicken sausages, frozen chicken meat balls (kofta), chicken shawerma and chicken mortedella, respectively using the conventional methods for isolation.Prevalence of L. monocytogenes in retail broiler chicken ready to eat meat products was (13.6%).
The demonstrated highest isolation rate among Listeria spp., were L. ivanovii (20.8%) and L. monocytogenes (13.6%) but, L. grayi (1.6%), L. seeligeri (0.8%) and L. welshimeri (1.2%) were of low presence in processed meat products.The present results are not in agreement with Awaisheh (2010) who found that the prevalence rate of isolated L. innocua and L. welshimeri were the most and least frequently isolated from 56 beef and 36 poultry samples.Osaili et al. (2011) reported that L. ivanovii were the predominant isolates among Listeria spp. in RTE chicken-shawerma samples (66.7%).RTE meat product samples were free of L. innocua.Listeria spp., distributed widely in the environment and vegetables, processed foods, silage and soil.These results could be explained by fecal contamination during evisceration, or to food handlers (Bresford et al., 2001).It appears that RTE products are generally subjected to potential cross-contamination during processing and handling.Therefore, it is generally accepted that efficient preventive measure has to be taken to limit the contamination of RTE products.
L. grayi, L. seeligeri and L. welshimeri isolates were recovered from raw chickens might enter the processing plant via the animals harboring Listeria spp., in the intestinal tract or as part of pharyngeal microflora.Incidence of Listeria spp., in RTE meat products could be attributed either to improper hygienic practice during processing or to food handlers Fenlon et al. (1996).These findings are also reported by Gibbons et al. (2006) who found Listeria spp., in (34.4%) of raw chicken and RTE meat products, in Trinidad.

CONCLUSION
In conclusion; this study reported the occurrence and distribution of L. monocytogenes and other Listeria species in retail meat products (frozen chicken burger, frozen chicken sausages, frozen chicken meat balls (kofta), chicken shawerma and chicken mortedella), purchased from restaurants in Khartoum state Sudan.The study recommended that the importance of hygienic conditions described in Sudanese HACCP program should be enforced in order to minimize presence of L. monocytogenes in raw dressed broiler chickens during manufacturing, handling and storage process at plant and retail stores level.

Table 1 :
Occurrence of Listeria spp. in retail broiler chicken ready to eat meat products in Khartoum State, Sudan Fig. 1: The isolation percentage of different types of Listeria spp., from 95 isolates of Listeria spp., from retail broiler chicken ready to eat meat products