PREVALENCE, VIRULENCE FACTOR GENES AND ANTIBIOTIC RESISTANCE OF BACILLUS CEREUS ISOLATED FROM READY TO EAT SANDWICHES

PREVALENCE, VIRULENCE FACTOR GENES AND ANTIBIOTIC RESISTANCE OF BACILLUS CEREUS ISOLATED FROM READY TO EAT SANDWICHES

Mohamed A. Hussein ;Ahmed E. Tharwatand Ayah M. Salem

Department of Food control, Faculty of Veterinary Medicine,

Zagazig University, Egypt

Key Words:Bacillus cereus, Sausage, Burger,  cereulide, Ready to eat, Sandwiches, Meat product.

ABSTRACT

A total of 105 random samples of ready – to – eat meat sandwiches were collected from different localities with different sanitation levels in Zagazig City. The collected samples were steak, kofta, burger,shawarma, hawawshi, liver and sausage (15 of each).The incidence of B. cereus was 40%, 100%, 86.66%, 46.66%, 33.33%, 66.66 % and 80% in examined steak, kofta, burger, shawarma, hawawshi, liver and sausage sandwiches, respectively. The  B. cereus counts were 2.73  ±  0.17, 4.16 ±  0.28, 3.98  ±  0.25, 3.17  ±  0.21, 2.84 ±  0.24, 3.34  ± 0.23and  3.57  ±  0.31log₁₀CFU/g in examined steak, kofta, burger, shawarma, hawawshi, liver and sausage sandwiches, respectively. The resistance of B.cereus was 100%, 90%, 90%, 80%, 75%, 75%, 70%, 70% and 65% for colistin, metronidazole, doxycycline, cephradine, streptomycin, thiamphenicol, cephaclor and erythromycin and ciprofloxacin, respectively.Meanwhile, the sensitivity obsereved for apramycin and kanamycin 55% and 45%,respectively. The multi antibiotic resistance (MAR) was ranged from 0.25 to 1. Six strain (30%) resist all examined antibiotics (12) moreover, 18 (90%) of B.cereus isolates considered as multi antibiotic resistant (resist three or more antibiotic from different classes). The cereulide(ces)gene  not detected,  The Non-hemorrhagic entero-toxin (nhe) gene detected in 7/10 (70%) and  cytotoxin (cytK) gene detected in 5/10 (50%) in identified B. cereus isolated from the examined ready to eat meat sandwiches.

1. INTRODUCTION:

Ready to eat foods are excellent concentrated nutrient sources which contain protein with a high digestibility score, essential amino acids, fatty acids, vitamins and minerals which are considered essential to optimal human growth and development. They also provide a source of readily available and nutritious meals for the consumers and are well appreciated by consumers because of their taste, low cost, nutrient value and ready availability for an immediate consumption. Various ready-to-eat (RTE) foods are becoming increasingly popular between high and low income people in developing countries where higher income people obtained RTE foods from restaurants and on the other hand low income people obtained RTE foods from street vendors on the street. The vendors congregate mainly in the central business district at major points of transit where large numbers of minibus taxi that are headed for different destinations. Foods are often held for several hours after cooking until sold, this included in some foods overnight at ambient temperature, although reheating could alleviate some hazards, but this action has not always been done effectively at vending site. Foodborne disease outbreaks linked with RTE foods have been associated with various foodborne pathogens (Gilbreth et al., 2005). The initial microbiological load on RTE food ingredients is important, however, factors such as handling, processing, storage and display may influence the microbiological load of RTE foods at the point of sale (Angelidis et al., 2006). 

Ready to eat meat products may be contaminated with microorganisms from meat handlers carrying of pathogenic bacteria during the processes of manufacturing, packaging and marketing. Improper cooking, refrigeration or storage may lead to meat borne illness. Foodborne pathogens are the leading causes of illness ranging from an upset stomach to more serious symptoms such as diarrhea, fever, vomiting, abdominal cramps and dehydration and death depending on the etiological agents (Van etal.,2007).

Foodborne illness is generally classified as an infection (ingestion of a harmful microorganism within a food. e.g. Salmonella andE.coli ,and intoxication (ingestion of a harmful toxin produced within a food. e.g. S. aureusor toxicoinfection (ingestion of a harmful microorganism within a food that produces a toxin in the human body, e.g. Bacillus cereus (Bean and Griffin, 1990).Antimicrobial resistance is a significant public health concern all over the world. The virulent and antibiotic-resistant bacteria cloud be transmitted to humans by the consumption of RTE meat sandwiches and the presence of these bacteria such as B. cereus, indicating poor hygienic measures which may produce a health risk for consumers (Gundogan et al., 2013) .

 The study was planned to determine prevalence, count, virulence genes antibiotic resistant pattern of B.cereus isolated ready to eat meat sandwiches.

2. MATERIALS AND METHODS:

2.1. Collection of samples:

 A total of 105 random samples of ready – to – eat meat sandwiches were collected from different localities with different sanitation levels in Zagazig City. The collected samples were steak, kofta, burger, shawarma, hawawshi, liver and sausage sandwiches (15 of each). All samples were directly transferred to the laboratory of Food Control Department in an ice box under hygienic conditions without undue delay then the core content were bacteriologically examined.

2.2. Bacillus cereus detection and identification:

The samples were prepared according to ISO 6887-2:(2003).Twenty five grams of each sandwiches core were homogenized aseptically for 1 min with 225 ml of 0.1 % peptone water in a stomacher (Colworth, 400) then serially dilutedto 10-fold in the same diluent.

The technique of Bacillus cereus count was performed according to ISO 7932:(2004). One hundred microlite from each prepared serial dilution of the samples under investigation was evenly spread over a dry surface of the B. cereus selective agar base with egg yolk and polymyxin supplement media. The plates  were inverted and incubated at 37 °C for 24 hours then examined for typical colonies of B. cereus which are turquoise blue 5 mm diameter with hallow zone of the same colour. The plates were re-incubated again for 24 hours before being counted againfor further growth. The number of suchcolonies were recorded as "presumptive" B. cereuscount.Isolated organisms were identified morphologically and biochemically.

2.3.Antibiogram for antibiotic sensitivity of isolated B.cereus:

All Bacillus cereus isolates were tested for their sensitivity to antibiotics by means of a disc diffusion method (Bauer et al., 1966). Sensitivity discs with variable concentrations were used to determine the susceptibility of the isolated strains (Oxoid Limited,Basingstoke, Hampshire, UK).                                                                     

      Agar plate method was applied by using of nutrient agar as a substrate for growth of the tested bacterium for its antibiotic sensitivity. The bacterial culture was uniformly spread on the surface of nutrient agar. Then the antibiotic discs were placed over the surface of inoculated plate. Moreover, the plate was then incubated at suitable temperature (25 °C) for 2-7 days and checked for the growth of the bacterium around the antibiotic discs. The maximal inhibition zone for the growth of microbe is said to that antibiotic had a maximum effect on the microbe growth.                        

       Therefore, the antimicrobial susceptibility testing was applied according to the guidelines stipulated by National Committee for Clinical Laboratory Standards "NCCLS" (2001). Accordingly, the antimicrobial discs and their concentrations as well as the diameters of the zones of inhibition for the tested strains are demonstrated in table (1): 

            The tested strains were evaluated as susceptible, intermediate and resistant.

Multiple Antibiotic Resistances (MAR) index for each strain was determined according to the formula stipulated by Singh et al. (2010) as follow:                                                                                                          

MAR index= No. of resistance (Isolates classified as intermediate were considered sensitive for MAR index) / Total No. of tested antibiotics.

2.4. Detection of emetic and diarrheic gene by using PCR:

The DNA of 10 isolated B.cereus strain extracted by QIAamp DNA Mini Kit,Catalogue no.51304 and the PCR Master Mix used for cPCRwasEmerald Amp GT PCR mastermix(2x premix).The Oligonucleotide primers used in cPCRMetabion (Germany) as shown in Tables (2& 3).

Table (1) Antimicrobial discs, concentration and interpretation of   their action on the isolated bacteria.

Table (2): Oligonucleotide primers sequences 

Table (3): Cycling conditions of the different primers   during cPCR

2.3. Statistical analyses:

The experimental data were evaluated using mixed model’s procedure, post hoc comparisons were applied, whenever appropriate, using Duncan’s test. All statistical procedures were performed using PASW statistics 18 (SPSS Inc., USA). Statisticalsignificance was considered at (P ≤ 0.05).

3. RESULTS AND DISCUSSION:

Farinaceous foods are the most common vehicles of the emetic type whereas the diarrheal type is associated with meat and soups (Kramer and Gilbert 1989).

The achieved results in table (4) declared that the incidence of B. cereus was 40%, 100%, 86.66%,46.66%,33.33% , 66.66 % and 80% in examined steak, kofta, burger, shawarma, hawawshi, liver and sausage sandwiches, respectively.

Table 4.  Prevalence and count log₁₀ CFU/g of B.cereus in comparison to standard of ready to eat meat (n= 105)

^{(a,b and c)} Means within the same column bearing different small superscript letters are significantly different (p< 0.05).   

  Min= Minimum              Max= Maximum                    SE= Standard Error

A lower incidence detected (37%) in the examined ready to eat foods collected from street vendors in Egypt detected by El-Sherbeeny et al. (1985),in Turkey 22.4% and 23.3% Guevenet al. (2006) and Büyükyörüket al. (2014), respectively.Found 28 % of meat samples positive for B. cereus. Willayatet al. (2007) and Das et al. (2009) found 23.5 % and 36.7 % contamination level of B. cereus, respectively. (20%) of B.cereus detected in RTE liver sandwiches samples by Abd-El-Malek (2014) This variation might be because hygienic practices followed in different localities in meat shops and restaurants.

Nearly similar incidence of B.cereus in examined marketed sandwiches (68%) was detected byEnan et al. (2012), (80%) in the examined chicken and meat products detected by Kamatet al. (1989). The results in table (4) showed that B. cereus counts were 2.73  ±  0.17, 4.16 ±  0.28, 3.98  ±  0.25, 3.17  ±  0.21, 2.84 ±  0.24, 3.34  ± 0.23and  3.57  ±  0.31log₁₀CFU/g in examined steak, kofta, burger, shawarma, hawawshi, liver and sausage sandwiches, respectively. The obtained counts were in the range of the previous studies by Konumaet al. (1988)whodetected the majority of the examined spices which ranged from to 2 to 4 log₁₀CFU/g, Rusul and Yaacob (1995) from 2.7-3.9 log₁₀CFU/g. TeGiffelet al. (1996) 2 to 6 log₁₀CFU/g in different food samples. Aksuet al. (2000) from 3 to4 log₁₀CFU/g. and Morshdy et al.,(2014) who found B.cereus counts were 2.5 x 10⁴ ± 6.3x10³, 3.9x 10⁴ ± 1x10⁴ and 3.3 x 10⁴ ± 7.6 x10³ CFU/g in examined kofta, liver and shawarma sandwiches, respectively.

There was a significant difference (p< 0.05) between examined samples which was attributed to the sandwich composition (plan with sauce or with sauce dressing and green vegetables), the additive introduced in processing of meat products, antimicrobial factors, environmental conditions, and potential differences in handling by personnel. According to Food Standards Australia New Zealand (2001) , data in table (4) revealed that 37 samples (35.3%) of the total examined samples were categorized as good < 10², while 14 (13.3%) of the total examined samples fell in the acceptable category (10² to <10³).

The unsatisfactory samples were 31 (29.5%) carry a significant risk if applied under certain conditions as excessive handling, bad hygienic conditions, and time and temperature abuse) that help B.cereus to proliferate and make it to fell in the category unsatisfactory. The presence of potentially hazardous level of B.cereus (>10⁴) in 23(21.9%) of total examined RTE sandwiches as recommended by Food Standards Australia New Zealand (2001).

    The presence of large numbers of B. cereus (more than 10⁴ CFU/g) in food is an indicative of active growth and proliferation of the organism and consistent with a potential hazard to health. It causes both diarrheal and emetic syndromes, each associated with distinct enterotoxin (Slabyj et al., 2003).

Toxin producing B. cereus plays an important role as the causative agent of two types of food poisoning: diarrhea and emesis. The emetic syndrome is mainly characterized by vomiting a few hours after ingestion of the contaminated food. In the diarrheal syndrome, symptoms appear 8–16h after ingestion, and include abdominal pain and diarrhea. In general, both types of food borne illness are relatively mild and self-limiting. Nevertheless, more severe cases have occasionally been reported involving hospitalization or even deaths (Lund et al., 2000; Dierick et al., 2005).

In the absence of control measures, virulent and antibiotic-resistant organisms might be transmitted to humans by the consumption of meat and their product and the presence of these organisms such as B.cereus in examined chicken meat products, indicating poor sanitary conditions which may create a health risk for consumers (Gundoganet al., 2006). Especially, multidrug resistant strains which are more dangerous and of great food safety concern (Van et al., 2012). As, the emergence of infectious disease caused by drug-resistant bacteria requires alternatives to conventional antibiotics and the search for new drugs is becoming critical because of the growing concern over the failing antibiotic drug discovery pipeline (Sulakvelidzeet al., 2001).

The data in figure (1) showed that the resistance of B.cereus was 100%, 90%, 90%, 80%, 75%, 75%, 70%, 70% and 65% forcolistin, metronidazole,doxycycline, cephradine, streptomycin ,thiamphenicol, cephaclor and erythromycin and ciprofloxacin,respectively.Meanwhile, the sensitivity obsereved for apramycin and kanamycin 55% and 45%, respectively.Antibiotic resistance of Bacillus cereus previously detected by Rusul and Jaacob (1995), Rahmati and Labbé (2008) and Ankolekaret al. (2009) for different types of antibiotics such as tetracycline, streptomycin and ceftriaxone.The data in table (5) showed that multi antibiotic resistance (MAR) was ranged from 0.25 to 1. Six strain (30%) resist all examined antibiotics (12) moresover, 18 (90%) of B.cereus isolates considered as multi antibiotic resistant (resist three of more antibiotic from different classes).The multidrug resistant Bacillus cereus is increased. It may be attributed to the use of antibiotics in animals raised for food for different purposes such as prophylaxis, and growth promotion, or therapeutics and these resistant bacteria can be transmitted to human through foods (Nygårdet al., 2008)Bacillus cereus strains are known to be frequently resistant to antibiotic therapy due to their capacity to produce an exopolysaccharide barrier (Gundogan et al., 2006), and carry a wide variety of multi –drug resistant genes on plasmids, which can be exchanged and spread among different species of bacillus (Neihartet al., 1988).

Figure (1): Antimicrobial susceptibilityof B. cereus isolated from examined from ready to eat sandwiches.

Table (5) Antimicrobial resistance profile of B. cereus isolated from examined from ready to eat sandwiches (N=20).

Colistin (CT)              Metronidazole (MTZ)      Doxycycline (DO)

Cephradine(CE)        Sterptomycin (S)               Thiamphenicol (TP)

Cephaclor (CEC)  Erythromycin (E)             Ciprofloxacin (CIP)

Norfloxacin (NOR)    Kanamycin (K)                  Apramycin (APR) No:number

A.R: antibiotics resisted by S.aureus

MAR: multi antibiotic resistant (No antibiotics resisted by B.cereus/  No examined antibiotics).

To compare the antibiotic resistance of the isolated B. cereusfrom foods in other studies the resistance of 50 randomly selected enterotoxin-positive isolates was determined using a panel of antibiotics previously employed in a study of B. cereus from fish (Rahmati and Labbé, 2008).Moreover, half or more of 50 isolates were resistant to three of the 10 antibiotics tested: ceftriaxone, streptomycin and tetracyclineAnkolekaret al. (2009). Resistance to tetracycline by foodborne B. cereus was observed previously (Rusul and Jaacob, 1995).

Thus, the antimicrobial susceptibility would have differed between each study. Thus, the rates of resistance to antibiotics of bacteria were high in developing countries, possibly as the result of the inappropriate or uncontrolled use of antibiotics with or without prescription (Chang et al., 2003).

Prevalence of virulent genes among isolated B.cereus strains isolated from the examined chicken meat products samples:

The spores of B.cereus survive normal cooking temperatures (Dierick et al., 2005) and proliferate when the additives stored at  room temperatures for long times; such temperature-abused can cause foodborne illness either by intoxication (emetic type) or infection (diarrheal type).Toxin producing Bacillus cereus plays an important role as the causative agent of two types of food poisoning: diarrhea and emesis. The emetic syndrome is mainly characterized by vomiting a few hours after ingestion of the contaminated food. In the diarrheal syndrome, symptoms appear 8–16h after ingestion, and include abdominal pain and diarrhea. In general, both types of food borne illness are relatively mild and self-limiting. Nevertheless, more severe cases have occasionally been reported involving hospitalization or even deaths (Lund et al., 2000 and Dierick et al., 2005).

• Egypt. J. of Appl. Sci., 34 (9) 2019                             247

  Cereulide(ces) gene:

The achieved results in Photograph (1) revealed that the (ces)gene  not detected in  identified B. cereus isolated from the examined ready to eat meat sandwiches.

Similar results revealed that the (ces) gene not detected during examination of B.cereus isolates by Chang et al. (2011)and  Hariram and Labbé  (2015) reported that none one of the 88 isolates obtained in their study possessed the emetic toxin (ces) gene.

Lower incidence of (ces) gene detected by Kimet al. (2011) found that  only nine from 68 sample of red pepper carrying the (ces) gene in comparison with  my study this result is considered to be low, Kimet al. (2015)  they detected only one emetogenic(ces) gene.

Cereulide(ces) gene causes vomiting, potentially by binding to the 5-HT3 receptors in the stomach/small intestine to stimulate the vagus nerve and brain (Agataet al., 1995). However, in recent studies, isolates harbouring emetic toxin genes were reported for B. cereus isolates from rice, spices, cooked rice and Sunsik and alsofarinaceous foods are known to be mainly associated with the emetic syndrome of B. cereus intoxications (Schoeni and Wong 2005).

• Non-hemorrhagic entero-toxin (nhe) gene:

The achieved results Photograph (1) revealed that the (nhe)gene detected in 7/10 (70%) in identified B. cereus isolated from the examined ready to eat meat sandwiches.

Photograph (1.)Agarose gel electrophoresis of multiplex PCR products amplified from genomoic DNA of Bacillus cereus.

The study coincide with the result obtained by TeGiffel et al. (1997)  they reported that (nhe) seem to be commonly distributed among B. cereus, in more than 50% of foodborne B. cereus isolates,  Andersen-Borge, et al. (2001) they found that  (nhe) genes were detected among most B. cereus strains tested in their study and Kim et al. (2011) they found (nhe)genes and were highly frequent among B. cereusstrains tested in this study in comparison with the other genes., which is in good agreement with previous reports that most isolates from different food samples contained the (nhe) genes.

cytotoxin K (cytK) gene:

The achieved results Photograph (1) revealed that the (cytK) gene detected in 5/10 (50%) in identified B. cereus isolated from the examined ready to eat meat sandwiches.

 The recently discovered cytotoxin K (cytK) was the cause of the symptoms in a severe outbreak of B. cereus food poisoning, which included production of bloody diarrhea in several people and three fatalities, in France in 1998 (Lund et al. 2000).The result is in line with the in previous work reported by Lund et al. (2000) who assumed that the occurrence of strains that possess only (cytK) is quite limited. Ehling-Schulz et al. (2006) they found that (cytK) gene was mainly found in combination with the two other enterotoxin genes, none of the tested isolates carried only (cytK).Higher prevalence was obtained byNgamwongsatit et al. (2008) they found (cytK)gene was frequently detected with higher incidence in the B. cereus with percentage of (88.81%),Chitovet al. (2008) who found (cytK) gene occurred in (70.4%) of the foodborne isolates tested. Generally, it was found that meat sandwiches from street vendors harbor B.cereus that has the ability to resist many antibiotics and contain harmful genes. Therefore, the executive authorities must intervene to protect consumers.

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مدى تواجد الجینات الضاریة ومقاومة المضادات الحیویة لعصیات سیرس المعزولة من سندوتشات اللحوم

محمد عبدالله محمد حسین، أحمد السید ثروت و أیه محمد على سالم

قسممراقبة الأغذیة - کلیةالطبالبیطری -جامعةالزقازیق- مصر

یزداد الاقبال علی منتجات اللحوم الجاهزة للأکل لدى الباعة الجائلینمن قبل المواطنین لسد جوعهم خاصة فئة العمال البسطاء، الذین یجدون أنفسهم مجبرون علی تناولها لتوافقها مع دخلهم الیومی،غیر مبالین اذا کانت صحیة ام لا. تباع هذة المنتجات دائما على عربات مصنوعة من الحدید فی أغلب الأحیان أو من الخشب، لها فاترینة زجاج حجمها دائما ما یکون صغیرا أو متوسطا لیقدر البائع منهم على جرها، بجوارها أسطوانة غاز لتحضیر الطعام علیها. تعتبرهذة المنتجات مصدرا من مصادر التسمم الغذائی وتشکل خطرا علی صحة المستهلک وذلک نتیجه لتعرضها للتلوث بالمیکروبات التی تؤدی الی فسادها قبل استهلاکها.تتواجد بکتریاعصیات سیرس بنسب بلغت 40٪ و 100٪ و 86.66٪ و 46.66٪ و 33.33٪ و 66.66٪ و 80٪. وکانت متوسطات أعدادها 2.73 ± 0.17 و 4.16 ± 0.28 و 3.98 ± 0.25 و 3.17 ± 0.21 و 2.84 ± 0.24 و 3.34 ± 0.23 و 3.57 ± 0.31 مستعمرة بکتیریة لوغاریتم ₁₀/ جرام من سندوتشات اللحم ؛ الکفتة ، البرجر  الشاورما؛ الحواوشی؛  الکبده والسجق. تم تصنیف 37 عینة (35.3٪) من إجمالی العینات التی تم فحصها على أنها جیدة تحتوى على أقل من²10 ، فی حین أن 14 (13.3٪) من إجمالی العینات التی تم فحصها کانت ضمن الفئة المقبولة (²10 إلى <³10). وکانت العینات غیر المرضیة 31 (29.5٪) ووجد أن نسبة العینات التى تحتوى علىأکثر من⁴10هى 23 (21.9٪) من إجمالی السندوتشات التى تم فحصها طقبا لأعداد عصیات سیرس. وکانت نسب مقاومة المضادات الحیویة لعصیات سیرس100% ، 90٪ ، 90٪ ، 80٪ ، 75٪ ، 75٪ ، 70٪ ، 70 ٪ و 65 ٪ للکولیستین ، المیترونیدازول ، الدوکسیسیکلین ، السیفرادین ، الستربتومایسین ، الثیامفینیکول ، السیفاکلور والاریثرومیسین و سیبروفلوکساسین ، على التوالی. ووجد أن العزلات کانت حساسة لکلا من أبرامیسین وکانامیسین بنسب 55 ٪ و 45 ٪ ، على التوالی. تراوح مؤشر المقاومة للمضادات الحیویة المتعددة لعصیات سیرس المعزولة من 0.25 إلى 1 ​​. علاوةعلىذلک،کانت 6 سلالات (30 ٪) متعددةالمقاومةلجمیعالمضاداتالحیویةالتیتماختبارهاو 18 (90 ٪) منسلالاتعصیاتسیرستعتبرمقاومةللعدیدللمضاداتالحیویة. وباستخدام تکنولوجیا تفاعل البلمرة المتسلسل لعشرة عزلات من عصیات سیرس لم یتم تحدید جین السیریولید بینما تم تحدید الجین المعوى الغیر نزیفى فى 7 (70%) والسیتوتوکسن جین فى 5(50%) من عصیات سیرس المعزولة من سندوتشات اللحوم من الباعة الجائلین.