PREVALENCE OF VIRULENCE GENES OF SOME FOODBORNE BACTERIA IN CHICKEN MEAT PRODUCTS

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

PREVALENCE OF VIRULENCE GENES OF SOME FOODBORNE BACTERIA IN CHICKEN MEAT PRODUCTS

NASHWA, M. ZAKI ¹; EL-DOSOKY. H.F.A¹; and WAFAA, M. GAD²

¹ Food Hygiene Dept. Mansoura Provential Lab, Animal Health Research Institute, Egypt

² Microbiololgy Dept. Mansoura Provential Lab, Animal Health Research Institute, Egypt

Received: 23 March 2017;       Accepted: 13 April2017

INTRODUCTION

Poultry meat and its products are very popular food throughout the world, it considered as cheap, good delicious and nutritious, source of protein with good flavour and easily digestion. Ready to eat food can be described as the status of food being ready for immediate consumption at the point of sale, it may be raw or cooked, and can be consumed without further treatment Tsang (2002). The importance of food as a vehicle for transmission of several diseases has been documented, especially in developing countries where the hygienic standards are not strictly followed or enforced Harakeh et al. (2005). Staph. aureus produces a wide variety of toxins including staphylococcal enterotoxins (SEs; SEA to SEE, SEG to SEI, SER to SET) with demonstrated emetic activity, SEs are a major cause of food poisoning, which typically occurs after ingestion of different foods, particularly chicken meat products, contaminated with Staph. aureus by improper handling and subsequent storage at elevated temperatures

Corresponding author: Dr. NASHWA, M. ZAKI

E-mail address: nashwazaki80@gmail.com

Present address: Food Hygiene Dept. Mansoura Provential Lab, Animal Health Research Institute, Egypt

Tharwat and Elabbasy (2014). Salmonellosis was one of the most commonly zoonotic disease accounting for 133,258 confirmed humancases Osek and Wieczorek (2010). Salmonella often present in fresh tissues due to defects during slaughtering process of poultry and carcass manipulation Lee et al. (1998) as well as Cebedo et al. (2008) concluded that S. spp. are pathogenic bacteria that can contaminate food products during or after processing. Hamilton et al. (2009) mentioned that E.coli was isolated from 60% of examined poultry from butcher shops with mean counts of 0.70 log₁₀ cfu/g. and 16% from poultry sold in supermarket samples with mean counts of 0.51 log₁₀ cfu/g. Matossian and Kingcott (1979) detected food poisoning outbreak from donar kebab (a product similar to shawerma). Staphylococcus spp., E.coli and S. spp. were isolated from raw chicken products and chicken shawerma Kaneko et al. (1999) and Pelczar et al., 2006). Shiga toxin (Stx)-producing E. coli (STX-EC), also known as Verotoxin-producing E. coli which associated with infantile diarrhea, haemorrhagic colitis, thrombocyticpurpura, and hemolytic uremic syndrome in humans Griffin and Tauxe (1991). The aim of this study was to assess the presence of these bacteria in some chicken meat products and the risk of contamination on the consumer.

MATERIALS AND METHODS

1- Collection of samples:

Two hundred samples of chilled chicken meat products (50 samples each of chicken luncheon, chicken burger, chicken sausage and chicken shawerma) at ± 4^o C were collected aseptically from different shops (small grocery and large supermarkets) from Mansoura city, Dakahlia province and transferred to the laboratory in an insulated ice-box without delay.

2- Bacteriological examination:

2.1- Preparation of food homogenate: according to technique recommended by ISO, 6887-2, (2003) 25 g. of each sample was removed by a sterile scissors and forceps and stomached using Seward stomacher 80 biomaster England with 225 ml sterile buffered peptone water (0.1%) to give a homogenate of 1/10 dilution from which ten fold serial dilutions were prepared and subjected to the following bacteriological examination.

2.2- Total E.coli count: according to technique recommended by FDA (2002a).

2.3- Staphylococcus aureus count: FDA (2002b). using Baird-Parker agar plates, incubated at 35 ^oC for 48 hr. The suspected Staphaureus colonies were isolated, purified and confirmed by coagulase test and the total count was calculated.

2.4- Isolation of E. coli according to technique recommended by ISO, 16649/2, (2001)

2.5- Isolation of Salmonellae ISO, 6579 (2002): by enrichment in peptone water at (37 ^oC for 24hr) then selection enrichment in Tetrathionate (37 ^oC for 24hr) and rappaportvasiliades at 41.5 ^oC for 18 hr., platting on XLD, MaCconkey's and Hektoneentreic agar at 37^oC for 24 hr. The presumptive colonies were confirmed biochemically and serologically.

3- Detection of virulence genes in Staphaureus, E. coli and Salmonella using PCR:

Carried out in Reference Lab for Quality Control on Poultry Production, Animal Health Research Institute, Dokki-Egypt.

3.1- DNA extraction:

DNA extraction from positive samples were(6 Staph. aureus), (4E. coli) and (1 Salmonella) performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56^OC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The samplewas then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

3.2- Oligonucleotide Primers:

The used Primers used were supplied from Metabion (Germany) are listed in Table (I) and Table (II).

3.3- PCR amplification:

For uniplex PCR, primers were utilized in a 25- µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentrations, 4.5 µl of water, and 6 µl of DNA template. For stx1, stx2 duplex PCR, primers were utilized in a 50- µl reaction containing 25 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 13 µl of water, and 8 µl of DNA template. The reaction was performed in an appliedbiosystem 2720.

3.4- Analysis of the PCR Products:

The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of the uniplex PCR products and 30 µl of the duplex PCR products were loaded in each gel slot. Generuler 100 bp ladder (Fermentas, Thermo Scientific, Germany) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

Table I: primer sequence for Staph. aureus enterotoxins genes used in multiplex PCR (Mehrotra et al., 2000).

Table II: Primers sequences, target genes, amplicon sizes and cycling conditions.

Statistical analysis:

The results are expressed as mean ± standard Error (SE). Data were statistically analyzed using statistical analysis systems. (SAS version 9.1, SAS Institute, Inc., 2003).

RESULTS

The achieved results of Staph. aureus in Tables (1 & 2) for Chicken luncheon, Chicken burger, Chicken sausage and Chicken shawerma were 3.2±1.6, 3.5±1.5, 3.6±1.4 and 3.7±1.3 log₁₀cfu/g. with incidence rate 6%, 2%, 2% and 2 %, respectively. The results of E. coli in Tables (1,2) for Chicken luncheon, Chicken burger, Chicken sausage and Chicken shawerma were 3.4±1.8, 3.1±1.3, 3.7±2.1 and 3.8±1.5 log₁₀ cfu/g. with incidence rate 2%, 4%, 0% and 2%, respectively, serologically the isolated E. coli indicates presence of the enterotoxigenic strains E. coli O127:H6 in chicken luncheon and E. coli O125:H21 and E. coli O127:H6 in chicken burger. Salmonella were not detected in Chicken luncheon, Chicken burger and Chicken sausage and detected in 2% of the examined chicken shawerma was S. typhimurium.

By PCR the results showed the presence of enterotoxin producing genes (A, C, D and E) in Staph. Aureus the three isolates of Staph. aureus isolated from luncheon showed the presence of  enterotoxin gene 1^st (A, E) , 2^nd (A, D) and 3^rd (B, D). The isolate of Staph. aureus isolated from burger showed presence of enterotoxin genes (A and E), the sausage isolate showed the presence of enterotoxin gene (A) while shawerma isolate showed presence of enterotoxin genes (A) .The virulence genes of shiga toxin (stx1 and stx2) were examined using PCR in the four E. coli isolates the results were postive for these genes.

Table1: Statistical analytical results of Staph. Aureus and E. coli in the examined samples expressed as log₁₀cfu/g.(n=50).

Table 2: The incidence, Serotyping and virulence gene of isolated, Staph. Aureus, E. coli and S. spp. from the examined samples (N= 50 of each).

ND. = not determined

No. = number of positive samples, C p =coagulase positive

DISCUSSION

Staph. aureus, E. coli and Salmonella are the major causes of food borne infection and intoxication and their presence in food conistitute an important hygienic problem for food processors, food handlers and consumers Bergadol (1989). The enterotoxication generally is not lethal and the elderly are more susceptible than the younger individuals, where the amount of STAPH. aureus enterotoxins required for intoxication about 94-184 ug Erol and Iseri, (2004).

The achieved results of Staph. aureus in Tables (1 & 2) for Chicken luncheon, Chicken burger, Chicken sausage and Chicken shawerma were 3.2±1.6, 3.5±1.5, 3.6±1.4 and 3.7±1.3 log₁₀cfu/g. with incidence rate 6%, 2%, 2% and 2 % respectively. The results nearly similar Saleh et al. (2010) who mentioned that Staph. Aureus count were 1.14x10³±3.32x10², 2.17x10³±4.31x10² and 2.2x10³± 4.45x10²/g. with different  incidence of 4%,12% and 16% for luncheon, beef-burger and sausage respectively higher percentage were reported by Amal, (2004) 15% and 25% in Staph. aureus for luncheon and fresh sausage; Fatin, (2004) could isolate Staph. Aureus from  luncheon in percentage of 16%; and Soultos et al. (2003) in percentage of 19.4% in luncheon; Mousa, (1993) reported that S. aureus count was 2.3x10⁴cfu/g. for luncheon.; Ahmed, (1992) 6.6% in sausage. EL-Mossalami et al. (2009) detected Staph. aureus in 92%, 80% and 88% with mean values of 3.25±6x10³, 2.8±1.4x10² and 4.1±2x10³cfu/g. in sausage, beef burger and shawerma respectively; Armany et al. (2016) could isolate S. aureus in percentage of24% and 20% in raw sausage and luncheon; Shawish and AL-Humam (2016) were 12%,22% and  30% in beef luncheon, beef burger and beef sausage; AL-Ghamdi, (2012) S. aureus count in chicken luncheon and chicken burger were 1.47x10⁶ and 1.2x10⁷cfu/g respectively. Ibrahim, (2009) detected Staph. aureus in 22.85% and 31.85% in luncheon, and sausage and EL-Khatieb (1997) (29%) in sausage. the percentage of coagulase positive Staph. aureus  strains isolated from Chicken luncheon, Chicken burger, Chicken sausage and Chicken shawerma were 6%,2%, 2%  and 2% respectively as in Table (2). Chomvarin et al. (2006); Oh, et al. (2007) and Chiang et al. (2008) concluded that the occurrence of enterotoxigenic Staph. Raureus in ready to eat food products has been reported in various parts all over the world; Shalaby and Zaki, (2008) could isolate 4, 5 and 3enterotoxigenic strains of Staph. aureus from beef burger, sausage and shawerma respectively and Motten et al. (2011) found Coagulase positive Staph. aureus in luncheon by 7%, 7% and 5% from the collected samples from three supermarkets. Eldaly et al. (2014) showed that the isolation percentages of Staph. aureus in the examined samples of luncheon, burger, and sausage were 15%, 10%, and 20% respectively.

The results of E. coli in Tables (1,2) for Chicken luncheon, Chicken burger, Chicken sausage and Chicken shawerma were 3.4±1.8, 3.1±1.3, 3.7±2.1 and 3.8±1.5 log₁₀ cfu/g. with incidence rate 2%,4%,0% and 2% respectively, serologically the isolated E. coli indicates presence of the enterotoxigenic strains E. coli O127:H6 in chicken luncheon and E. coli O125:H21 and E. coli O127:H6 in chicken burger. These results nearly similar to Samaha et al. (2012) were 8% in chicken luncheon; Ibrahim (2009) were 5.71% in luncheon; Fawzy (2004) were 8% in luncheon and Amal (2004) were 5 and 25% in luncheon and fresh sausage. Meanwhile, higher results were recorded by Armany et al. (2016) 20% and 24% in raw sausage and luncheon respectively and Mousa (1993) were14% in luncheon; Ibrahim, (2009) were 42.85% in sausage and Fathi et al. (1992) in luncheon and sausage which were 41.67% and 20%which may be due to post processing contamination or unefficient cooking and improper handling.

Salmonella were not detected in Chicken luncheon, Chicken burger and Chicken sausage and detected in 2% of the examined chicken shawerma was S. typhimurium. EL Jakee et al. (2014) detect Salmonella in burger, sausage and poultry products by 10, 35 and 25% respectively, the isolated Salmonella were S. enteritidis and S. typhimurium and Samaha et al. (2012) could isolate  8% Salmonella in chicken luncheon, Amal (2004); Ibrahim (2009) and Saleh et al. (2010) can not find Salmonella in luncheon while in sausage Mousa (1993); Saleh et al. (2010); Kozacinski et al. (2008); Ibrahim (2009); and Tudor et al. (2010) can^'t found S. spp. in fermented sausage. Amal (2004) found salmonella by 5% in sausage. The health hazard from Salmonella must not be underestimated. The fact that Salmonella was detected in samples from supermarkets, where chicken are displayed under refrigeration, shows that the spread of infection was not only confined to seemingly unhygienic environments FAO, (2013). It was suggested that to prevent contamination by Salmonella control measures must be taken at all stages of the food chain, from agricultural production, to processing, manufacturing and preparation of foods in both commercial establishments and at home WHO (2013).  

PCR was applied to evaluate the presence of virulence genes in the isolated Staph. aureus, E. coli and Salmonella. Staph. aureus is one causes of food poisoning, its pathogenicity result from possession of virulence genes that produce different toxins which result in self-limiting sever illness. For this reason the virulence genes of 6 isolated coagulase positive Staph. aureus were examined by PCR and the results  showed the presence of enterotoxin producing genes (A, C, D and E) in Staph. aureus the three isolates of Staph. aureus isolated from luncheon showed the presence of  enterotoxin gene 1^st(A, E) , 2^nd (A, D) and 3^rd (B, D). The isolate of Staph. aureus isolated from  burger  showed presence of  enterotoxin  genes (A and E), the sausage  isolate showed the presence of enterotoxin gene (A) while shawerma isolate showed presence of enterotoxin genes(A) as shown in Table (I) (Photo No. 1). The result agree with Eldalyet al. (2014) who found that luncheon samples harbored seb gene s while burger samples harbored sed gene also Tharwat and Elabbasy (2014) reported that SEA enterotoxin gene was the predominant enterotoxin genes which were detected in examined chicken burger and chicken luncheon.

Staph. aureus enterotoxin were analyzed from ready to eat products including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat in a study conducted by Fijalkowski et al. (2016), this study reported that the most prevalent enterotoxin genes were sei (36%), seln (32%) and encoding exfoliative toxin A (37%). Another study conducted by Puah et al. (2016) revealed an incidence of (96.2%) virulence genes from Staph. aureus isolated from 200 food samples. A total of 30.8% of the isolates carried SE gene which cause food poisoning meanwhile the most common enterotoxin genes found were seg (11.5%) and egc (5.8%). On the other hands Inv A and stn virulence genes in the isolated S. Typhimurium were positive. InvA gene was amplified and detected at 284 bp while stn gene detected and amplified at 617 bp. In Korea, Li et al. (2006) could detect 17 virulence genes from isolated Salmonella using PCR assays, 14 genes assayed (82.4%) out of these 17 genes included invA gene.

The virulence genes of shiga toxin (stx1 and stx2) were examined using PCR in the four E. coli isolates the results were positive for these genes Table (2) (Photo No. 2), these results were nearly similar to Balague et al. (2006) who collected 500 food samples from shops selling ready to eat foods in Argentina and E. coli virulence gens were examined by multiplex PCR (stx1, stx2, eae A, cnf1, cnf2, ein v, Lt1, ST1 and ST11), ten E. coli isolates showed  the presence of stx1, stx2 genes while other genes were negative. Another study carried by Bohaychuck et al. (2006) reported shiga toxin producing E. coli O22: H8 from beef samples in Alberta and Canada.

Photo No. (1): Agarose gel electrophoresis of Staph. aureus PCR products using enterotoxins Staphylococcus primer.

Lane "1": 100 bp DNA ladder

Lane "2 ": positive amplification of 102 bp for enterotoxin A, 209 bp for enterotoxin E, 278 bp for enterotoxin D and 451 bp for enterotoxin C

Lane "3": positive amplification of 102 bp for enterotoxin A, 209 bp for enterotoxin E, 278 bp for enterotoxin D and 451 bp for enterotoxin C

Lane "4": positive amplification of 102 bp for enterotoxin A and 278 bp for enterotoxin D

Lane "5": positive amplification of 102 bp for enterotoxin A

Lane "6": positive amplification of 102 bp for enterotoxin A

Lane "7": posistive amplification of 164 bp for enterotoxin B and 278 bp for enterotoxin D

Photo No. (2): Agarose gel electrophoresis of and E. coli PCR products using stx1 and stx2 primers.

Lane "1": 100 bp DNA ladder

Lane "2 ": positive amplification of\614 bp for stx1 gene and 779 bp for stx2.

Lane "3": positive amplification of 779 bp for stx2.                 

Lane "4": positive amplification of \614 bp for stx1 gene and 779 bp for stx2.

Lane "5": positive amplification of 779 bp for stx2.

Photo No. (3): Agarose gel electrophoresis of Salmonella and PCR products using invA, and stn, primers

L= 100 bp DNA ladder.

N= negative control.

P= positive control (give amplificationat 617pb for stn gene, 284 bp for invA, 614 bp

Sample of S.Typhimurium showed 284 bp amplification for invA gene and 617 pb for stn gene.

CONCLUSION

This study confirms that chicken meat products may serve as a source of foodborne pathogens and accordingly a potential public health hazard. Corrective action needs to be employed to minimize the risk of consuming this type of fast food, such action must aim to minimizing the bacterial contamination during the production of chicken meat products (cleaning, cutting, seasoning and stacking), its cooking and serving. Regular surveillance by the public health regulatory bodies will ensure compliance with WHO and ISO standards for food safety.

Also, handling, storage and processing steps are major avenue for the cross contamination of the major materials used for the preparation of such product. Personal hygiene and processing practice of the food vendors are major factors.

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