Assiut University web-site: www.aun.edu.eg

DETECTION OF MULTI DRUG RESISTANT FOOD BORN BACTERIA IN             READY TO EAT CHICKEN MEAT

ZEINAB AHMED ¹ AND  SHIMAA EL- NAGAR ²

¹Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP), Animal

 Health Research Institute, Agricultural Research Center, Luxor, Egypt., Dokki, Giza

² Reference Laboratory for Veterinary Quality Control on

Poultry Production (RLQP), Animal Health Research Institute, Agricultural Research Center, Luxor, Egypt

Received: 19 December 2020;     Accepted: 20 January 2021

INTRODUCTION

Foodborne illness is major health problem associated with ready to eat chicken

Corresponding author: Zeinab Ahmed

E-mail address: zeinabrenad@gmail.com

Present address: Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP), Animal Health Research Institute, Luxor, Egypt

meat (Tabashsum et al., 2013).  Multidrug resistant food borne microorganisms made the food safety situation more vulnerable in public health (Ahmed et al., 2011). Contamination of ready-to-eat foods persists through preparation and cooking due to the quality of raw materials (Dhama et al., 2013). Staphylococcus spp. contamination of excessive handling during preparation of the meal (Mensah et al., 2002). Foodborne illness is one of the significant public health problems worldwide Hereford, microbiological safety of food has become an important concern for consumers, various industries, and regulatory agencies (Bavisetty et al., 2018). Some microorganisms maintain their normal life functions in food and are used in food production, whereas others may cause food spoilage or foodborne diseases. as Salmonella spp., Campylobacter spp., Staphylococcus aureus, Escherichia coli and Shigella spp. (Saglam and Seker 2016). Approximately 1.8 million people died due to food-borne diarrheal infections each year in developing countries (WHO, 2007).Using of raw ingredients, insufficient workers hygiene, holding for long period, lead to contamination of food with pathogenic microorganisms (Gundogan et al., 2005). E. coli has become recognized as a serious foodborne pathogen that associated with diseases outbreaks including diarrhea, hemorrhagic colitis and the life-threatening hemolytic uremic syndrome in humans (Hussein, 2007). Consumption of ready-to-eat products is thought to be the major cause of the Salmonella outbreaks (Thai et al., 2012). Shigella dysentriae contaminated water, food, and unhygienic environment like overpopulated area, malnourished people, and poor waste management area, usually it survives poorly outside human body (Wilson et al., 2005). Mould and mycotoxin contamination of food by feed ingredients (Trenholm et al., 2000). Bacteria and fungi are associated with Chicken meat spoilage acquired during slaughter, packaging, transportation or selling and handling include species of Salmonella, Shigella, Staphylococcus, Escherichia coli, Campylobacter, Penicillium and Cladosporin (Clarence et al., 2009). Antibiotics are used for control and treatment of bacterial diseases in poultry. There is growing scientific evidence that use of antibiotics in food animals leads to the development of resistant pathogenic bacteria that can reach humans through the food chain (Van Looveren et al., 2001). Recent reports have shown that different types of food and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in human or veterinary medicine and in food-producing animals (Anderson et al., 2003). Annual cost of treating infections caused by antibiotic-resistant bacteria is estimated to be $4 to $5 billion (McGowan 2001). Therefore, the present study was carried out for determination of APC, Coliform count  & Staphylococcus counts , Anaerobic count, Yeast count, Mould count and identification of St.aureus, entero pathogenicE. coli, Salmonella, Shigella for ready to chicken eat meat meals including chicken shawirma, chicken burger without addition, and fried chicken 

MATERIAL AND METHODS 

1. Collection and transportation of samples:                                                                    

A total of 150 random samples of ready to eat chicken  meat meals including, chicken shawirma, chicken burger each of them without addition, and fried chicken.  (50 of each of product) were collected from different restaurants in Luxor City. Each sample was kept in a separate sterile plastic bag, put in an ice box then transferred to the laboratory under complete aseptic conditions without undue delay for bacteriological examinationof samples.

2. Preparation of samples (APHA 1992):                                                              

Twenty five grams of the samples were taken under aseptic condition to sterile stomacher bag then 225 ml sterile Buffer peptone water were added, the contents were homogenized at Stomacher for 2minutes, the mixture was allowed to settled, for 5 minutes at room temperature. The contents were transferred into sterile flask, thoroughly mixed, 1 ml was transferred into separate sterile test tube containing 9 ml sterile slain, from which ten fold serial dilutions were prepared. 

3. Isolation and identification of food-borne bacteria:                                 

The prepared samples were subjected to the following bacteriological examination: Determination of Aerobic Plate Count using ISO 4833-1:2013. Coliform count using ISO 4832\2006. Determination of total anaerobic bacterial count by Roberts et al., 1995. Determinations of Staphylococci countusing ISO 6888-1-1999. Enumeration of yeast and Mould at 25˚C for 5-7 days using ISO 21527-2:2008. Isolation of E.coli by ISO, 2001 and Varnam-Evans, 1991. The method for detection of S.aureus by ISO 6888-1-1999. The method for detection of Salmonella by ISO 65 79, 2002. The method for detection of Shigella usingISO 21567: 2004  

4. Identified serologically by using diagnostic sera:

 Serological diagnosis of Salmonella according to Kauffman – white scheme – 1974 for determination of somatic antigen(O) and Flagler antigen(H) using DENKA  SEIKEN CO®Japan). )Sero diagnosis of E.coli according to                                                                                                

5. Antibiotic

sensitivity:                                    
Antibiotic sensitivity tests were done by using disc diffusion test following the method described by (WHO and CDC 2002). Were interpreted with the standard diameters of (NCCLS 2002) and were recorded as sensitive (S), intermediate (I) and resistant (R). The following antibiotics will be used for disc diffusion test: Antibiotics will be used in Salmonella, E.coli, ShigellaAmoxicillin-Streptomycin-Gentamycin-Nalidixic-Colistin sulphat - Ceftriaxaon Ciprofloxacin- Neomycin- Sulphamethazol. Antibiotic used in Staph. Ampicillin- Clindamycin- Oxacillin -Erythromycin- Gentamicin- Vancomycin- Tetracycline-Chloramphenicol.

RESULTS

Table 1: Statistical analytical results of shawirma without addition in the examined samples (n=50).

Table 2: Statistical analytical results of burger without addition in the examined samples (n=50).     

Table 3: Statistical analytical results of fried chicken in the examined samples (n=50).

Table 4: The incidence of Staph, E. coli and Salmonella in samples.

Table 5: Serological identification of entero pathogenicE.coli in Samples.

Table 6: Serological identification of Salmonella in Samples.

Table 7: Show the result of sensitivity test for Salmonella spp.

Most isolate are sensitive for these antibiotic Gentamycin (10ug), Colistin sulphate (10ug)and Ceftriaxaon but Streptomycin is(R)and Nalidixic (I)

Table 8: Show the result of sensitivity test for E.coli in Samples.

Most of E.coli isolate sensitive for Colistin sulphate(10ug) and Nalidixic acid(10ug)

Table 9: Show the result of sensitivity test for Staphylococcus aureus in Samples.

Most of isolate are sensitive to Ampicillin AM (10ug), Erythromycin (15 ug) and    Vancomycin VA (15ug) and Gentamycin, Clindamycin and Chloramphenicol are (R)

DISCUSSION

The total aerobic plate count is important for evaluation of sanitary condition of ready to eat chicken meat. Limits suggested for total aerobic bacterial count in ready to eat chicken meat range not less than 10 ⁴ microbes /g (ISO 4833-1:2013). Accordingly the high bacterial count of some examined samples may be attributed to neglected sanitary measures during their handling, preparation and serving. The mean values of APC for chicken shawirma without addition, burger without addition and fried chicken were 1.36X10² (cfu/g), 5.58X10³(cfu/g) and 4.62X10³(cfu/g).These results were Nearly (El Taher-Amna 2009) who found that APC 5.38´10³ (cfu/g) from burger. The mean values of Coliform for chicken shawirma without addition, burger without addition and fried chicken were 8.42X10² (cfu/g), 3.34X10² (cfu/g), 1.53X10² (cfu/g). The current results lower than  (El-Rayes 2008) who found that the mean value of Coliform was 3.8´10³/g in  ready to eat chicken meat and agree with (Saad et al., 2011) who found that the mean value of Coliform was 5.17´10² in the examined samples. The mean values of Staph Count for chicken shawirma without addition, burger without addition and fried chicken were 4.67 (cfu/g), 2.33 (cfu/g), 1.33 (cfu/g). The low incidence of Staph.aureus attributed to exposure of those products to high temperature during processing (Amal, 2004). The mean values of Yeast and Mould for chicken shawirma without addition, burger without addition and fried chicken were 7.67, 2.67 (cfu/g) and 8.33, 2.67 (cfu/g) and 4.33, 1.67 (cfu/g). respectively Mould and yeast contamination usually occurred due to handling, deboning, processing, packing and washing with polluted water and may due to dust, flies, air, workers, equipment and fluctuation of temperature during transportation and storage(Farghaly, 1998). Salmonella is responsible for disease in humans. It has been the leading cause of many outbreaks and infections around the world and is considered as one of the major causes of human gastroenteritis (Rasschaert et al., 2005).In these result isolates were 6 (4%) Salmonella from 150 examined ready to eat chicken meat samples. The serological examination of identified Salmonella isolates showed that S. typhimurium, S. enteritidis, S. Anatum and S.Kentucky were detected. These results was higher than (Al-Mutairi 2011)and less than( Shaltout et al., 2013) and (Harakeh et al., 2005)  which reported prevalence of Salmonella 7.4 %.  Most isolate of Salmonellae are sensitive for Gentamycin (10ug), Colistin sulphate (10ug), Ceftriaxone which agree with (Gomba et al., 2016), (Lamas et al., 2016) and (Arora et al., 2015) and dis agree with (Stopforth et al., 2006) who said most of isolate of Salmonellae are resist for Ceftriaxone. The presence of Staph aureus in heat treated food may be due to its contamination from food handlers, inadequate cleaned equipment or post processing contamination (Duffy et al., 2000). 8(5.33%) isolates of St.aureus were isolated from 150 examined these results was higher than those obtained by (Shafizi et al., 2016) who recorded Staph. aureus was 2.3% and  less than (Ali-Sohaila and Abd-Elaziz-Doaa, 2011) who recorded Staph. aureus was  30% and agree with, (Diaz-Lopes et al., 2011) who recorded 6.3% of Staph. aureus. Cooking plays a great role in killing of most of these microorganisms but not all. Presences of heat resistance toxins from some of these bacteria represent a great public health hazard especially in places with great groups of people receiving this food. Also, post cooking recontamination when holding of such meals for a period until serving in unhygienic condition especially at room temperature or insufficient reheating represent of major public health hazard. Food handlers are important source of Staphylococcal Spp for food contamination in restaurants and food outlets (Colombari et al., 2007). As a result there is an increased risk of pathogens surviving and transferring not only by cross-contamination, but also by undercooking as observed in this kind of fast-food industry (Nimri-Laila et al., 2014). Most of isolate sensitive for Colistin sulphate and Nalidixic acid. The result agree with (Abdul Matin et al., 2017). E.coli play an important role as human pathogens, which give rise to gastroenteritis outbreaks, severe diarrhea in infants, coli-bacillosis in adults, meningitis and enteritis (Youssef et al., 1992). 9(6%) isolates of E.coli were isolated from 150 examined ready to eat chicken meat samples. O125:H2, O143:H8 and O111:H1 from chicken shawirma without addition, O55: H2, O158:H3, O86a:H1 from chicken burger without addition, O142:H3, O26:H1. Isolates were positive for fried chicken. These results agreement with (Mohammed et al., 2014), O55:H2 these results nearly in agreement with, (DíazSánchez et al. 2012). Most  isolate  of E.coli  were sensitive for Colistin sulphate (10ug) which agree with (El-Sukhon  et al., 2002) and dis agree with (Obeng et al., 2012) who said most of isolate  of E.coli  are resist for Nalidixic acid.  

CONCLUSION  

This study indicated that RTE Chicken meats are often contaminated with APC, Coliform, Staphylococcus, Yeast, Mould Counts and Staph. aureus, Salmonella  and E.coli. There is need for the relevant local authorities to ensure that the food sold to consumers in fast food restaurants is safe, wholesome and fit for human consumption in order to prevent outbreaks of food-borne illnesses. Addition of vegetables lead to increase contamination and count of microorganism, so the samples without addition of vegetables low in contamination and count of microorganism, Shawirma is more contaminated product because low in cooking. Cooking plays a great role in killing of most of these microorganisms but not all. After Cooking may occur recontamination when holding of such meals for a period until serving in unhygienic condition especially at room temperature or insufficient reheating represent of major public health hazard. Food handlers, Utensils, bad Quality packaging and bad environment are important source ofthecontamination There should be In addition Procurement of raw materials of the best possible microbiological quality, Prevention of undue contamination of fast foods prior to processing, Regular training to food handlers in all aspects of food hygiene and safety, Quality packaging to keep foods fresh and get rid of health risk factors, Adequate storage, ideal transportation, and hygienic handling to finished product, Microwave oven treatment prior to serving to consumption, Ensuring good quality raw materials, adequate lethality treatment, and effective sanitation of both the equipment and processing environment.

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