OCCURRENCE AND MOLECULAR TYPING OF CLOSTRIDIUM PERFRINGENS USING VIRULENCE GENE IN SOME COOKED READY- TO-EAT MEAT AND POULTRY PRODUCTS IN

Dept. of Food Hygiene,Faculty of  Veterinary Medicine,

AssiutUniversity.

Occurrence and molecular typing of Clostridium Perfringens using Virulence Gene in Some Cooked Ready- To-Eat Meat and Poultry Products in

Assiut City, Egypt

(With 3 Tables and 2 Figures)

By

Doaa M. Abd El-Aziz; M.A. Mohamed*

and Sohaila Fathi Hassan Ali**

Dept. of Poultry Diseases, Faculty of  Veterinary Medicine,

AssiutUniversity

Dept. of Food Hygiene, Animal Health Research Institute, Assiut Regional Laboratory, Egypt.

(Received at 15/3/2011)

مدى تواجد والتصنيف الجزيئى لللکلوستريديم برفرنجنز بإستخدام جين الضراوة فى منتجات اللحوم والدواجن المطهية والجاهزة

فى مدينة أسيوط – مصر

دعاء محمد عبد العزيز ، مؤمن عبد العظيم محمد ، سهيله فتحى حسن على

ميکروب الکلوستريديم برفرنجنزالمسبب للأمراض المعوية، هذا الميکروب ينتج بويغات بعضها مقاوم للحرارة، لذلک تم تحليل بعض منتجات اللحوم والدواجن المطهية الجاهزة للتعرف على تواجد الميکروب من عدمه. تم عد الخلايا فى منتجات اللحوم والدواجن الجاهزة 80 عينة بواقع 20 عينة من کل من الکفتة، شاورمة اللحمة، الفراخ المشوية وشاورمة الفراخ. وجد أن العدد فى معظم العينات کان أقل من 10 مستعمرة / جرام (88.7 %). أيضا تم تحليل العينات بإستخدام إختبار البلمرة المتعدد والثنائى للتعرف على جينات التسمم وأوضحت الفراخ المشوية أنها الأعلى فى معدل تواجد هذا الميکروب بنسبة 40%، کما أن الجين المسبب للتسمم الغذائى وجد فيها بنسبة 20%. وکان نسبة العزل فى الکفتة وشاورمة الفراخ للميکروب هى 20% و5% بالترتيب ولم يوجد فى شاورمة اللحمة بعد التأکد بإستخدام إختبار البلمرة.

Summary

Clostridium perfringens is spore former microorganism and some spores are heat-resist, so some cooked ready-to-eat meat and poultry products were analyzed for its presence. C.perfringens vegetative cells were counted in ready-to-eat (RTE) meat and poultry products (80 samples; 20 of each kofta, meat shawrma, grilled poultry and poultry shawrma) and found that most (88.7%) of the examined samples indicated < 10 CFU/ gm. The organism was isolated on Tryptose sulphite cycloserine agar with egg yolk (TSC with egg yolk), then the examined samples were analyzed by using multiplex and duplex PCR for detection and genotyping of C.perfringens toxin genes. Grilled poultry showed the highest incidence of C.perfringens (40%) by using PCR and Type A producing enterotoxin (cpe gene) which is responsible for food poisoning could be detected at rate of 20%. The incidence in kofta and poultry shawrma was 20% and 5%, respectively, but in meat shawrma after confirmation with PCR the organism failed to be detected.

Key words: C. perfringens, Toxin genes, Ready-To-Eat.

Introduction

C. perfringens grows well on RTE meat and poultry products due to absence of oxygen. C. perfringens is ubiquitous in the environment, also in raw meat either vegetative or spore form. RTE meat and poultry products contaminated with high levels of certain strains of C. perfringens vegetative cells may lead to food poisoning. Vegetative form is killed by heating, but spore form not. The acidic conditions encountered upon passage through the gastrointestinal tract trigger the sporulation of vegetative cells (Wrigley et al., 1995).

C. perfringens is a gram-positive, spore-forming, nonmotile, obligately anaerobic, encapsulated rod. The heat sensitivity of                   C. perfringens spores being defined as not surviving 100°C for 60 min. Four toxins, alpha, beta, epsilon, and iota, are used for classifying all strains into five types (A–E) depending on which combination of these four toxins a given strain produces. Most cases of food poisoning due to          C. perfringens are due to type A that produces only the alpha toxin, which is distinct from CPE (cpe) that is also produced. The toxin is released when cells lyse following spore formation and acts on epithelial cells causing diarrhea and loss of water and ions. The alpha toxin (cpa) is considered the main lethal toxin of C. perfringens and is a multifunctional phospholipase causing hydrolysis of membrane phospholipids resulting in cell lysis. Food poisoning due to C. perfringens usually occurs 8–24 hr after ingestion of food containing large numbers of vegetative cells. Diarrhea and severe abdominal pain usually occur (Levin, 2010).

Berry and Gilbert (1991)stated that production of enterotoxin occurred during sporulation in intestine and McClane (1997) reported that the organism can grow and multiply rapidly after germination, so that cooked meat and poultry products must be cooled rapidly to restrict germination, outgrowth, and vegetation. Enterotoxin production (CPE) is associated with sporulating cells. Pre-formed CPE in foods is often not implicated in foodborne illness, as heating for 5 min at 60°C will inactivate the enterotoxin. Petit et al. (1999) cleared that the toxinogenic typing A, B, C, D or E of C. perfringens is not based upon the serologic specificity of CPE-related food borne illness, but many other exotoxins produced by the microorganism and designated α, β, ε, and ι. Type A strains produce only α -toxin, type B α -, β - and ε-toxin, type C α - and β -toxin, type D α - and ε-toxin, and type E α - and ι -toxin. The different toxin types cause different forms of enteritis and enterotoxaemia in various hosts (Songer, 1996). Various PCR protocols including multiplex PCR assays have been established to genotype C. perfringens isolates with respect to the genes cpa, cpb, etx, iap, cpe and cpb2, encoding the α -, β -, ε-, ι -, entero- and β₂ -toxin, respectively (Meer and Songer, 1997 and Kadra et al., 1999).

The type A food poisoning is the major virulence factor in the common form of food poisoning due to the production of the enterotoxin (CPE). CPE is produced in the small intestine after ingestion of at least 10⁷ C. perfringens cells. C. perfringens type C food poisoning is rare. If not treated, the disease is often fatal and has a mortality rate of 15–25% even with treatment. These toxins are all produced during the vegetative growth of C. perfringens type C(Brynestad and Granum, 2002).

C. perfringens spores that germinate after heat treatment, and ultimately contaminate the RTE product, depends on such factors as the time-temperature factor (Crouch and Golden, 2005).

The aim of the work was to detect density and amount of               C. perfringens in RTE meat and poultry products, also to genotyping the isolated strains by multiplex and duplex PCR because there is few data about studies on incidence of C. perfringens in RTE meat and poultry products. 

Materials and Methods

Samples:

Eighty samples, 20 each of kofta, meat shawrma, grilled poultry and poultry shawrma collected from several restaurants in Assiut city were used for isolation of C. perfringens. The samples were transmitted rapidly to the laboratory under sterile condition.

Counting of C. perfringens:

Preparation of the samples:

Ten grams of each sample were weighted and thoroughly mixed with 90 ml of 0.1% peptone water.

Decimal dilutions were prepared from the previous dilution. 1 ml from each dilution was placed and mixed with 15 ml of melted Tryptose sulphite cycloserine agar with egg yolk (TSCE) into sterile Petri dishes. After solidification, the solidified agar was overlaid with further 10 ml from of Tryptose sulphite cycloserine agar without egg yolk (TSC) agar and incubated anaerobically at 37°C for 20±2h. All blackish colonies were counted and recorded (Robert and Greenwood, 2003).

Isolation of C. perfringens

Preparation of the sample:

10 g of the sample were homogenized with 10 ml of Thioglycollate medium as enrichment and incubated anaerobically at 37°C for 20±2h.

Loopfuls were streaked on Tryptose sulphite cycloserine agar with egg yolk (TSC with egg yolk), and incubated anaerobically at 37°C for 20±2h. Colonies thought to be C. perfringens appear black due to reduction of sulfite and with halo zone due to lethicinase enzyme. Colonies were confirmed by Gram stain, streaking on blood agar (β-haemolytic colonies with double zone of haemolysis), motility, nitrate and lactose-gelatin medium (Wen and McClane, 2004).

PCR assay for detection and genotyping of C. perfringens isolates.

1- DNA extraction: 

Two or three colonies from each isolate grown on reinforced clostridial agar were suspended in 300 µl distilled water, and the mixture was then incubated at 56°C for 30 minutes. The samples were treated with 300 µl of TNES buffer (20 mM Tris pH 8.0, 150mM NaCl, 10mM EDTA, 0.2% SDS) and proteinase K (20 mg/ml). After incubation at 37°C for 2 h the mixture was boiled for 10 minutes. To that suspension, same volume of phenol (saturated with Tris-HCL) was added; the suspension was shaken vigorously by hand and centrifuged at 1000 rpm for 10 minutes. The upper phase was transferred into another tube and sodium acetate (0.1 volumes) and ethanol (2.5 volumes) were added. The suspension was kept at –20°C for 1.5 h and than centrifuged at 11000 rpm for 10 minutes. The pellet was washed with 95 and 70% ethanol, each step followed by 5 min centrifugation. Finally the pellet was dried and resuspended in 50 µl distilled water.

2- PCR assay:

Polymerase chain reaction analysis was performed on two reactions: the first is multiplex PCR for detection of genes encoding alpha- toxin (cpa), beta-toxin (cpb), epsilon-toxin (etx), and the second is duplex PCR for detection of iota-toxin (cpi)and enterotoxin (cpe)genes. PCR primers and fragment length arelisted in Table 1.

PCRs were performed in 50 µl mixture containing 25 µl GoTaq Green Master Mix (Promega, Madison, WI), 0.5 µl of each primer (20 mM), 4µl whole–cell DNA as template and molecular DNase-RNase free water (Promega, Madison, WI) prepared to complete a final reaction volume of 50 µl. The thermal cycling conditions were as follows: initial denaturation for 4 min at 95°C followed by 35 cycles of 1 min at 95°C, 1 min at 60°C for the first multiplex and 56°C for the second, 1 min 20 sec at 72°C and a final step of 2 min at 72°C.      

PCR products were separated by electrophoresis on 2% agarose gel (Sigma) added with 0.5 µg/ml ethidium bromide (Sigma). Gels were visualized under UV transilluminator and the image was captured using GelLogic 100 imaging System (Kodak).

Table 1: Toxin genes, oligonucleotide primer sequences and length of amplification products of the C. perfringens multiplex and duplex PCR (Drigo et al., 2008):

Results

Table 2: Frequency distribution of the examined ready-to-eat meat and poultry products based on their C. perfringens count.   

Table 3: Incidence of C. perfringens in the examined meat products (20 of each) by using multiplex and duplex PCR.

   Type A: cpa gene                                            

   Type A and cpe positive i.e producing enterotoxin(cpe): cpa + cpe genes

   Type C: cpa + cpb     

Figure 1: Amplification of C. perfringens toxin genes by multiplex and duplex PCR. Lane 1,2,3,4,5 and 6 C. perfringens typeA (α toxin gene (cpa) amplification); lane 1e, 2e, 3e, 5e and 6e                     C. perfringens type A producing enterotoxin (cpe amplification).

        N.B. the same DNA is used in two lanes; one in multiplex and the other in duplex PCR reaction as shown in Table 1.

Figure 2: Amplification of C. perfringens toxin genes by multiplex and duplex PCR. Lane 7, 8, 10,12, 13 and 14cpa gene amplification; lane 10e cpe gene amplification; lane 11 C. perfringens type C     (cpa and cpb gene amplification).

Discussion

Meat and poultry products are generally implicated in most outbreaks, with beef products responsible for about 40% of C. perfringens foodborne outbreaks (Bhunia, 2008). Roast beef is a major vehicle of outbreaks because of improper handling, temperature abuse, and inadequate cooling after cooking. The Centers for Disease Control (CDC) estimated that 248,520 foodborne illnesses due to C. perfringens occur in the USA annually (Mead et al., 1999).

The maximum counts of colonies suspected to be C. perfringens in kofta, meat shawrma ranged from 10⁴- <10⁵ , while the maximum counts in grilled poultry and poultry shawrma ranged from 10²- <10³ CFU/g, respectively (Table 2). Most of the examined samples (88.7%) showed count of <10/g. Kalinowski et al. (2003) found that the majority of the examined cooked ready-to-eat meat and poultry samples (79%) indicated < 10 CFU/ g. However, 20% of the samples showed > 10 CFU/g and 6% had >100 CFU/g. This indicated that the samples contained heat resistant spores of the pathogen.Another study reported that all the tested meat and poultry samples contained very low number (<10) of C. perfringens(Skariyachan et al., 2010). Various studies indicated higher counts of       C. perfringens; 7.6-log10 (Juneja et al., 2001) in cooked cured beef; 8.07-log10 (Juneja and Marks, 2002) in cooked cured chicken; and 8.03-log10 (Huang, 2003) in cooked ground beef.

On January 6, 1999, Food Safety and Inspection Service (FSIS) published a final rule in the Federal Register (FSIS Docket No. 95-033F; 64 FR 732) establishing performance standards for C. perfringens in cooked beef, roast beef, and cooked corned beef products; fully and partially cooked meat patties; and certain fully and partially cooked poultry products, in an effort to address the public health risk posed by                 C. perfringens. The production requirements for these products included performance standards limiting multiplication of C. perfringens to a maximum of 1-log10 within the product during RTE food manufacture(Crouch and Golden, 2005).

As shown in Table 3, Figure 1 and 2, the incidence of                    C. perfringens in kofta was 20%, 15% of the samples were type A positive and 5% of the examined kofta samples were type A producing enterotoxin. The prevelance of the pathogen in grilled poultry was 40%, 15% of them were type A positive, 20% were type A producing enterotoxin and 5% were type C. Poultry shawrma showed incidence of 5% which producing enterotoxin. All the examined meat shawrma showed negative result for    C. perfringens. Cpetx and cpi toxin genes are not indicated in all the examined samples. Based on the above results grilled poultry showed the highest incidence of C. perfringens which may be due to contamination of the processing plant and processed carcasses of broiler chickens with       C. perfringens, primarily from intestinal contents of poultry during the slaughter process via cross contamination (Akmak et al., 2006).

The above results were nearly in agreement with Wen and McClane (2004) who found that56 (38%) of 147 retail chicken samples in America, 24 strain were type A positive and one strain type A producing enterotoxin (cpe). 25 (23%) out of 108 samples of retail ground beef were contaminated with C. perfringens, 23 strain were type A positive, but non cpe positive. Skariyachan et al. (2010)found C. perfringens at a rate of 20% (2/10) in cooked meat samples and 8% (1/12) in cooked chicken samples. Also C. perfringens was isolated at incidence of 23% from cooked beef samples in a study performed by Kouassi et al. (2011).

In conclusion C. perfringens may cause food poisoning through consumption of RTE products especially poultry products. Its presence due to post heat treatment contamination of commodities is possible and may occur through handling, slicing and air transmission.(Ray, 1996).  Improper cold storage of RTE meat and poultry products accounts for approximately 90% of the predicted C. perfringens foodborne illness. Outbreak observations suggest that heavily spiced foods, such as some Mexican style foods, may be a contributing factor to C. perfringens outbreaks (Crouch and Golden, 2005).

References

Akmak, Ö.; Ormanci, F.S.B.; Tayfur, M. and Erol, Ü. (2006): Presence and contamination level of Clostridium perfringens in raw frozen ground poultry and poultry burgers. Turk. J. Vet. Anim. Sci., 30: 101-105.

Berry, P.R. and Gilbert, R.J. (1991): Clostridium perfringens type A food poisoning. In: Duerden, B.I., Draser, B.S. (Eds.), An-aerobes in Human Disease, London, Arnold, pp. 362–371.

Brynestad, S. and Granum, P.E. (2002): Clostridium perfringens and foodborne infections. Int. J. Food Microbiol., 74: 195–202.

Bhunia, A.K. (2008): Foodborne microbial pathogens-mechanisms and pathogenesis (pp. 158–159). New York: Springer.

Crouch, E. and Golden, N.J. (2005):A Risk assesment for C. perfringens in ready-to-eat and partially cooked meat and poultry products. USDA, Food Safety Inspection Service                                                                             http://www.fsis.usda.gov/PDF/CPerfringens_Risk_Assess_Sep2005.pdf.

Drigo, I.; Agnoletti, F.; Bacchin, C.; Bettini, F.; Cocchi, M.; Ferro, T.; Marcon, B. and Bano, L. (2008):Toxin genotyping of Clostridium perfringens field strains isolated from healthy and diseased chickens.Ital. J. Anim. Sci., 7:397-400.

Huang, L. (2003): Dynamic computer simulation of Clostridium perfringens growth in cooked ground beef. Int. J. Food Microbiol., 2716: 1–11.

Juneja, V.K.; Novak, J.S.; Marks, H.M. and Gombas, D.E. (2001): Growth of Clostridium perfringens from spore inocula in cooked cured beef: Development of a predictive model. Innovative Food Science and Emerging Technologies,2: 289–301.

Juneja, V.K. and Marks, H.M. (2002): Predictive model for growth of Clostridium perfringens during cooling of cooked cured chicken. Food Microbiol.,19: 313–317.

Kadra, B.; Gouillou, J.P.; Popoff, M. and Bourlioux, P. (1999): Typing of sheep clinical isolates and identification of enterotoxigenic Clostridium perfringens strains by classical methods and by polymerase chain reaction (PCR). FEMS Immunol. Med. Microbiol., 24: 259–266.

Kalinowski, R.M.; Tompkin, R.B.; Bodnaruk, P.W. and Pruett, W.P. (2003): Impact of cooking, cooling and subsequent refrigeration on the growth or survival of Clostridium perfringens in cooked meat and poultry products. J. Food Prot., 66: 1227–1232.

Kouassi, K.A.; Dadie, A.T.; Nanga, Z.Y.; Dje, K.M. and Loukou, Y.G. (2011): Prevalence of sulfite reducing Clostridium species in barbecued meat in Abidjan, Côte d’Ivoire. J. Appl. Biosci.,     2518–2522.

Levin, R.E. (2010):Rapid detection and characterization of foodborne pathogens by molecular Techniques.Chapter 14, P.: 487-522.

McClane, B.A. (1997): Clostridium perfringens. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington, DC. P.: 305– 326.

Mead, P.S.; Vance Dietz, L.S.; McCaig, L.F.; Bresee, J.S.; Shapiro, C.M. and Griffin, P.M. (1999): Food-related illness and death in the United States. Emerg. Infect. Dis., 5: 607–625.

Meer, D.R. and Songer, J.G. (1997): Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. Am. J. Vet. Res., 58: 702– 705.

Petit, L.; Gibert, M. and Popoff, M.R. (1999): Clostridium perfringens toxinotype and genotype. Trends Microbiol., 7: 104–110.

Ray, B. (1996): Fresh and ready-to-eat meat products. In Fundamental Food Microbiology. CRC Press Inc. pp. 214–218.

Robert, D. and Greenwood, M. (2003):Isolation and enrichment of microorganisms. In Practical Food, 3^rd Ed., Section 6, P. 146-149. Blackwell publishing.

Skariyachan, S.; Mahajanakatti, B.S.; Biradar, B.U.; Sharma, N. and Abhilash, M. (2010):Isolation, identification and charachterization of clostridium perfringens from cooked meat-poultry samples and in Silico biomodelling of its delta enterotoxin. International Journal of Pharmaceutical Sciences Review and Research. P: 164-172.    

Songer, J.G. (1996): Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev., 9: 216–234.

Wen, Q. and McClane, B. (2004): Detection of enterotoxigenic Clostridium perfringens type A isolates in american retail foods. Appl. Environ. Microbiol., p. 2685–2691.

Wrigley, D.M.; Hanwella, H.D.S.H. and Thon, B.L. (1995): Acid exposure enhances sporulation of certain strains of Clostridium perfringens. Anaerobe, 1: 263-267.