This study presents a survey of the microbiological quality of turkey meat sold in various outlets in Meknes city of Morocco and examines antimicrobial resistance of Staphylococcus aureus and Escherichia coli strains isolated to warn customers about the emergence of food poisoning. 96 samples randomly taken on different outlets, including 24 at the popular market, 24 at artisanal slaughterhouses, 24 at poulterers’ shops and 24 at supermarket. According to the microbiological criteria, 83.3% of samples did not meet the standards for E. coli. 95.8%, 33.3%, 41.6%, 41.6% of the samples purchased from supermarket, poulterers’ shops, artisanal slaughterhouses and popular market outlets, respectively, showed satisfactory quality point of view S. aureus among which 8.3% (8/96) of samples could be linked to a foodborne due to a concentration of S. aureus upper in 5 log10 υfc/g. The level of contamination E. coli and S. aureus at supermarket was recorded significantly lower (p<0.05) compared to other sites.
Among the 40 E. coli tested, the highest resistance was to amoxicillin-clavulanic acid (80%), followed by norfloxacin (67.5%), cephalothin (65%), nalidixic acid (62.5%), ampicillin (52%), trimethoprim/sulphamethoxazole (42.5%), ciprofloxacin (40%), cefoxitin (35%), ceftazidime (32.5%) and amikacin (15%). Low resistance rates were returned (between 5 and 12.5%) for ertapenem, aztreonam and gentamicin. For S. aureus, the highest percentage of resistance was found to the following antimicrobial agents: teicoplanin (67.5%), tetracyclin (40%) and vancomycin (30%). No resistance to the rest of antibiotics was found.
The bacterial load present on the surface of poultry carcasses reflects the general hygiene conditions in which they are prepared, stored, transported and sold. These data revealed also that the E. coli and S. aureus isolates recovered from the retail turkey meats were resistant to multiple antimicrobials, which can be transmitted to humans through food products.
Keywords: Turkey meat; Microbiological quality; Morocco; Health; Antimicrobial resistance
Food borne diseases and poisoning are the widespread and great public health concerns of the modern world. Both developed and developing countries are largely affected by food borne infections. Food borne diseases not only affect people’s health and well-being, but also have economic impacts on individuals and the countries , while the impact in case of developing countries is higher. It reduces markedly social and economic productivity of the countries . Because of the relatively high frequency of contamination of poultry with pathogenic bacteria, raw poultry products are reported to be responsible for significant number of cases of human food poisoning .
Poultry meat contributes substantially to the human diet . In Morocco, poultry meat is an important, low cost source of animal protein. This encourages the consumption of poultry products by a large number of consumers. Poultry meat is increasingly used by the growing rural and urban populations. This explains the high production in Morocco especially that the turkey fell from 10.5 to 50 million tonnes in recent years . However, foods of animal origin, especially poultry and meat, are the major vehicles for the transmission of human salmonellosis due to cross-contamination events or inadequate cooking . In Morocco, although several efforts diseases have been made to improve food safety and quality, food borne diseases still represent one of the main causes of mortality .
Amongst the food borne pathogens, Salmonella and S. aureus are the most common and frequent pathogens responsible for food poisoning and food related infections [8,9]. According to WHO , there was 25% diarrhea in foodborne illness caused by food infected with E. coli. In Morocco, Salmonella, S. aureus and Clostridium perfringens are reported to cause 42.8, 37, and 1.7% of food poisoning, respectively .
Four kinds of turkey meat outlets are used in Morocco: popular market, artisanal slaughterhouses, poulterers’ shops and supermarket. They differ from each other by the level of hygiene, diet cold which is subject carcasses (ambient temperatures, refrigeration, freezing). At popular market and artisanal slaughterhouses the conditions of slaughter and sale of the product are faulty , indeed turkey is slaughtered and scalded in hot water. After that, the carcasses are plucked and eviscerated mostly by hand. Before and after evisceration, broil carcasses are subject to washing and other operations which my disseminate bacteria from localized sites to the rest of the carcass as well as among carcasses. This kind of poultry is often sold in parts and the selling can take time, during which the carcasses are displayed at ambient temperatures during the day and put in the refrigerator for the night [12,13]. In these shops, the conditions are favorable to potential contamination by pathogens which may originate from the animal itself and environment factors (water, litter, air). On the contrary, poulterers’ shops and supermarket are an automated poultry slaughtering process established recently, whereby automated systems are used for scalding, plucking, eviscerating and packaging carcasses. Carcasses are then stored at 4°C before sale to supermarkets and Poulterers’ shops. These shops ensure the storage and sale of poultry meat under good hygienic conditions .
Besides, Morocco is a developing country with abuse of antibiotics in animal husbandry and it may cause antimicrobial resistance of bacteria animals. Schroeder et al.  proved that antibiotic resistance of bacteria isolated from humans was transferred from antibiotic resistant bacteria in animal.
The purpose of this study is to detect the contamination bacteria of retail turkey meat in retail markets in Meknes (Morocco) and examined antimicrobial resistance of S. aureus and E. coli strains isolated to warn customers about the emergence of food poisoning.
Between October 2011 and October 2012, a total of Ninety-six samples of turkey breasts with skin were collected from retailers, of which 24 samples were from popular market, 24 from artisanal slaughterhouses, 24 from poulterers’ shops and 24 from a supermarket in Meknes (centre-south Morocco). Each sample was placed in a separate sterile plastic bag. Samples were transported to the laboratory immediately after collection in an ice chest and tested upon arrival or stored at 2°C for no longer than 4h.
All bacterial counts were expressed as Log10 colony forming unit per g (Log10 CFU/g). To compare the Log10 values of microbial counts, the data were analyzed using Student’s t-test for each type of microorganism. Significance was determined at the 5% level.
A 25 g sample of skin was taken aseptically by scalpel excision and stomached in a sterile stomacher bag containing 225 ml of peptone water (Biokar Diagnostics, France) for 2 min. Decimal dilutions were carried out using the same diluents.
Mesophiles were determined using plate count agar (Oxoid, England) spread plates incubated at 30°C for 72 h. S. aureus on Baird- Parker agar with egg yolk-potassium tellurite emulsion plates (Bio- Rad), incubated at 35 ± 1°C for 24 to 48 h and typical colonies (black surrounded by clear zones) were tested for coagulase activity using rabbit plasma (Biokar Diagnostics, France) after activation by overnight incubation in Brain Heart broth (Biokar Diagnostics, France) at 35°C. E. coli counts, on rapid’ E. coli Agar (Bio-Rad, France) incubated at 37°C for 18 to 24 h, typical E. coli were considered as violet-to-pink.
Susceptibility to antimicrobials
Antibiotic susceptibility testing was performed by a disc diffusion method on Mueller-Hinton agar. The categories susceptible or resistant were assigned on the basis of the critical points recommended by the French committee on guidelines for susceptibility testing .The strains were screened for their resistance to the following antibiotics (Marnes-La-Coquette, France): nalidixic acid Na 30 μg; ciprofloxacin CIP 5 μg; ceftazidime CAZ 30 μg; amoxicillin-clavulanic acid AMC 20+10 μg; cefoxitin FOX 30 μg; cefotaxime CTX 30 μg; lincomycin, MY 15 μg; fusidic acid FD 10 μg; tetracycline TE 30 UI; teicoplanin TEC 30 μg; gentamycin CN 15 μg; vancomycin VA 30 μg; Rifampycin RD 30 μg; amikacin AK 30 μg; ertapenem ETP 10 μg; cephalothin KF 30 μg; aztreonam ATM 30 μg; ampicillin AM 10 μg; trimethoprim/ sulphamethoxazole SXT 1.25/23.75 μg and norfloxacin NOR 5 μg. We used the Automated System (OSIRIS) for reading and interpreting results (Bio-Rad).
It is important to determine the aerobic total criterion which is used as hygienic indicator in the slaughter- process. In total of 96 samples, the number of bacteria was 4.15 Log10 CFU/g in minimum registered in samples from the supermarket and in maximum 8.66 Log10 CFU/g registered in samples from slaughterhouses (Table 1). From this point of view parameter (mesophiles), the percentage of unacceptable samples was 48.95% (Table 1). This result was higher than the result obtained by Cohen et al.  in Morocco with 29.2% unacceptable samples of poultry meat poultry. In Hanoi, Nguyen Van Ton  found 54.65% meat poultry samples over national standards (Figure 1a).
|Mesophiles||E. coli||S. aureus|
|Samples from||Log10 ufc/g||N%||Log10 ufc/g||N%||Log10 ufc/g||N%|
|Super market||4.15-7.60||19/24||2.65- 4.78||5/24||0- 4.48||23/24|
|Popular market||4.88-8.26||15/24||1.67- 4.96||4/24||2.3- 5||10/24|
|Range*||<6.7 Log10 ufc/g||<2.2 Log10 ufc/g||<3.7 Log10 ufc/g|
Table 1: Bacterial counts (Log10 cfu/g) found in retail turkey outlets (n=96).
Coli form bacteria are indicator organisms as enterobacteriaceae are of intestinal origin. Indicator organisms may be employed to reflect the microbiological quality of foods relative to product shelf life or their safety from food borne pathogens. Microbial indicators are more often employed to assess food safety and sanitation than quality [19,20]. According to WHO , there was 25% diarrhea in food borne illness caused by food infected with E. coli. In this study, for these bacteria, average counts were 3.43 Log10 CFU/g in samples purchased from super market and 3.85 Log10 CFU/g in those purchased from artisanal slaughterhouses (Figure 1c). In this study 100% of food samples were infected with E. coli. There was 5.49 Log10 CFU/g in maximum and 1.67 Log10 CFU/g in minimum (Table 1). Proportion of unacceptable sample was 83.3% (Table 1), higher than some results announced. Tran Thi Hanh et al  found 68.75% samples of poultry meat sold in Hanoi’s market. In America, 38.7% meat poultry samples in Washington infected with E. coli (Cuiwei Zhao et al.) . Cohen N et al.  indicated 48.4% samples infected with E.coli in Morocco, 22.4% among these was unacceptable. Levels of contamination of samples were significantly (P<0.05) higher in poulterers’ shops, slaughterhouses and in popular market than in supermarkets, possibly due to the good hygienic conditions in the supermarkets at the time of the previous stages (Figure 1c).
S. aureus has long been recognized as one of the food poisoning bacteria of concern to human health worldwide . Average counts of S. aureus in samples purchased from popular market and artisanal slaughterhouses outlets were 3.85 and 4.19 Log10 CFU/g respectively (Figure 1b). These levels of contamination were higher to those obtained by Álvarez-Astorga et al.  in chicken legs. Similarly, Waldroup AL  obtained values ranging from 3 to 5 Log10 CFU/g. Counts found by other authors are very variable. However, in samples purchased from supermarket, S. aureus counts were lower than those of other outlets. On the basis of the CNERNA-CNRS guidelines , 95.8%, 33.3%, 41.6%, 41.6% of the samples purchased from supermarket, poulterers’ shops, artisanal slaughterhouses and popular market outlets, respectively, showed satisfactory quality. The rest of the samples were considered of the unacceptable quality (Table 1). Enumeration of S. aureus revealed that the count of pathogen exceeded 5 Log10 CFU/g in 8 out of the 96 analysed samples (8.3%). Such high level of contaminated with S. aureus has been associated with increased risk for staphylococcal food poisoning . High contamination of food with S. aureus has been related to improper personal hygiene of employees during handling and processing .
This comparison should be made with caution because several factors must be taken into account when making such comparisons, including differences in country and origin, type of meat samples, sampling seasons, slaughterhouse sanitation, and isolation methods.
Food is an important factor for the transfer of antibiotic resistances. Such transfer can occur by means of antibiotic residues in food, through the transfer of resistant food-borne pathogens or through the ingestion of resistant strains of the original food microflora and resistance transfer to pathogenic microorganisms [29,30]. S. aureus strains are known to be frequently resistant to antibiotic therapy due to their capacity to produce an exopolysaccharide barrier and because of their location within micro abscesses, which limit the action of drugs .
Antibiotic resistance in S. aureus strains to 8 antimicrobial agents is shown in Table 2. Overall, the highest percentage of resistance was found to the following antimicrobial agents: teicoplanin (67.5%), tetracyclin (40%) and vancomycin (30%). No resistance to the rest of antibiotics was found. Otalu et al.  reported 100% resistance in S. aureus isolates from poultry meat against tetracycline and 61.5% against methicillin in Nigeria . Multidrug resistant S. aureus have been reported several times . Extensive uses of these antibiotics are thought to be the major cause of drug resistance in food borne pathogens .
|Antibiotic||Concentration Disc||S (Sensitive)||R (Resistant)|
|diameter (mm)||n||%||diameter (mm)||n||%|
|FOX||30 µg||≥ 27||40||100||<25||0||0|
|MY||15 µg||≥ 22||40||100||<17||0||0|
|FD||10 µg||≥ 24||40||100||<24||0||0|
|TE||30 UI||≥ 23||24||60||<21||16||40|
|TEC||30 µg||≥ 17||13||32.5||-||27||67.5|
|CN||15 µg||≥ 20||40||100||<13||0||0|
|VA||30 µg||≥ 17||28||70||-||12||30|
|RD||30 µg||≥ 29||40||100||<24||0||0|
Table 2: Result of antibiotic resistance of S. aureus isolated.
Antibiotic resistance in E. coli strains to 14 antimicrobial agents is shown in Table 3. Overall, the highest percentage of resistance was found to the following antimicrobial agents: amoxicillin-clavulanic acid (80%), norfloxacin (67.5%), cephalothin (65%), nalidixic acid (62.5%), ampicillin (52%), trimethoprim/sulphamethoxazole (42.5%), ciprofloxacin (40%), cefoxitin (35%), ceftazidime (32.5%) and amikacin (15%). Low resistance rates were returned (between 5 and 12.5%) for ertapenem, aztreonam and gentamycin. In Morocco, Chaiba et al.  obtained resistance in E. coli strains of poultry meat to tetracyclin (80%), chloramphenicol (6.6%), amoxiline (20%), acid nalidixic (26.6%), gentamycin (0%), Neomycin (6.6%) and trimethoprim/ sulphamethoxazole (33.33%). Antunes et al.  also reported a high antimicrobial resistance of Salmonella isolates recovered from poultry products including chicken and turkey to nalidixic acid, tetracycline and streptomycin (ranging from 36% to 50%) but low resistance rate to trimethoprim (3%) in Portugal. We noticed also an increase of resistance to ciprofloxacin (40%), that represent the treatment of choice of severe non typhoidal Salmonella infection in adults [36,37]. This could be related to the use of some fluoroquinolones form methaphylactic or therapeutic purposes in poultry feed and drinking water.
|Antibiotic||Concentration Disc||S (Sensitive)||R (Resistant)|
|diameter (mm)||n||%||diameter (mm)||n||%|
|NA||30 µg||≥ 20||15||37,5||<15||25||62,5|
|CN||15 µg||≥ 18||38||95||<16||2||5|
|CIP||5 µg||≥ 25||24||60||<22||16||40|
|AK||30 µg||≥ 17||34||85||<15||6||15|
|FOX||30 µg||≥ 22||26||65||<15||14||35|
|ETP||10 µg||≥ 28||35||87,5||<26||5||12,5|
|CTX||30 µg||≥ 26||40||100||<23||0||0|
|CAZ||30 µg||≥ 26||17||42,5||<19||13||32,5|
|KF||30 µg||≥ 18||14||35||<12||26||65|
|AMC||20/10 µg||≥ 21||8||20||<16||32||80|
|ATM||30 µg||≥ 27||32||80||<21||2||5|
|AM||10 µg||≥ 19||19||47,5||<16||21||52,5|
|NOR||5 µg||≥ 25||13||32,5||<22||27||67,5|
Table 3: Result of antibiotic resistance of E. coli isolated.
The results of this study indicate the lack of unsatisfactory sanitary conditions and quality control during manufacturing and/or post production handling of the turkey meats, and a possible health safety problem. Also, the results clearly indicate that attempts have to improve the sanitary conditions in traditional turkey meat production procedure and we recommend more restrictions on the irrational use of antibiotics and public awareness activities should be undertaken to alert the public to the risks of the unnecessary use of antibiotics.