Detection of virulence genes of enterotoxigenic Staphylococcus aureus isolated from chicken meat cuts by using PCR

Please download to get full document.

View again

of 8
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Similar Documents
Information Report
Category:

Self-Help

Published:

Views: 0 | Pages: 8

Extension: PDF | Download: 0

Share
Description
The present study was performed on 120 random samples of fresh chicken meat cuts of Drumstick, thigh and breast (40 of each) which were purchased from different supermarkets and retail chicken butchers at Kaliobia Governorate, for detection of
Tags
Transcript
  410 Detection of virulence genes of enterotoxigenic  Staphylococcus aureus isolated from chicken meat cuts by using PCR Fatin S. Hassanen 1 , Fahem A. Shaltout 1 , Amani, M. S. 1 , Maarouf, A. A. A. 2 , Rasha, N. A 2   1  Food hygiene Dept., Faculty of Veterinary Medicine, Benha University. 2  Animal Health Research  Institute, Benha Branch  –   Food Hygiene Dept. A B S T R A C T The present study was performed on 120 random samples of fresh chicken meat cuts of Drumstick, thigh and breast (40 of each) which were purchased from different supermarkets and retail chicken  butchers at Kaliobia Governorate, for detection of virulence genes responsible for Staphylococcus. aureus  enterotoxins production by using PCR.A total of 33 examined samples were positive for S.aureus  counts represented as 13(32.5%) from drumstick samples; 11(27.5%) from thigh samples and 9(22.5%) from breast samples. The results revealed that 8 strains out of 10 random examined strains  by SET- RPLA test were enterotoxigenic and classified according to type of toxin into (3A;1C,2D and 2A&C).Moreover, PCR results cleared that ; sea gene was detected in 4 studied strains;  seb gene in one strain and  sec ;  sed   and  see  genes were detected in 3 strains out of 10 studied strains. Keywords :   Chicken meat, enterotoxigenic   S.aureus , virulence genes   ( http://www.bvmj.bu.edu.eg )  ( BVMJ- 33(   2):   410-417 ,   DECEMBER,   2017 )   1.   INTRODUCTION Chicken meat is a nutritious, healthy food that is low in fat and cholesterol compared to other meats but an excellent source of  protein. On the other hand, it is an ideal medium for bacterial growth because of high moisture content, richness in nitrogenous compounds (essential amino acids, proteins), good source of minerals, vitamins and other growth factors (Prange  et al  ., 2005). Chicken meat is a common vehicle of Staphylococci mainly Staph. aureus  that considered as one of the most important causes of foodborne outbreaks in people (Losito et al., 2005). Staphylococcus aureus  is the most  pathogenic species that is considered the third-most important cause of food-borne disease in the world (Liu et al., 2006 and  Normanno et al., 2007) and has two aggravating characteristics, toxin  production and antimicrobial resistance S. aureus  produce disease when the bacteria contaminate food, produce some enzymes which are implicated with staphylococcus invasiveness and many extracellular substances some of which are heat stable enterotoxins that renders the food dangerous even though it appears normal and extensive cooking can killed the  bacteria but the toxins may not be destroyed because most of them are gene  based i.e. they can be carried on the  plasmid (Prescott et al., 2005). S. aureus   produces a wide variety of toxins including Staphylococcal enterotoxins (SEs; SEA to SEE, SEG to SEI, SER to SET) with demonstrated emetic activity, and Staphylococcal-like (SEl) proteins, which are not emetic in a primate model (SElL and SElQ) or have yet to be tested (SElJ, SElK, SElM to SElP, SElU, SElU2 and SElV). SEs and SEls have been traditionally subdivided into classical BENHA   VETERINARY   MEDICAL   JOURNAL   V OL .   33,   N O .   2:   410-417,   DECEMBER,   2017 BENH UNIVERSITY F CULTY OF VETERIN RY MEDICINE   Hassanen et al. (2017)   .   BVMJ,   33,    N O .   2:   410-417   411 (SEA to SEE) and new (SEG to SElU2) types. The enterotoxin genes are accessory genetic elements in S. aureus , meaning that not all strains of this organism are enterotoxin producing (María et al., 2010). The Staphylococcal enterotoxins (SEs) are responsible for the symptoms that associated with Staphylococcal food  poisoning (Lieweiyn and Cohen, 2002). The disease is characterized by rapid onset symptoms including nausea, violent vomiting, abdominal cramps and diarrhea lasting from 24 to 48 h and the complete recovery usually occurs within 1-3 days. The illness is usually self-limiting and only occasionally it is severe enough to warrant hospitalization. Moreover,  sea  is the most common enterotoxin recovered from food  poisoning outbreaks (Pinchuk et al., 2010; María et al., 2010 and Shijia et al., 2016). RPLA (Reverse Passive Latex Agglutination) can identify enterotoxins using specific antibodies for each of the enterotoxins, but cross reaction between SEA and SEE have been reported (Sergeev et al., 2004). Several reports have described the development of polymerase chain reaction technique (PCR) protocols for the detection of SEs genes as it is not require  bacterial enrichment before detection of a specific gene (Mehrotra et al., 2000 and Sharma et al., 2000). So, the present study was performed for studying virulence genes enterotoxin production of   S. aureus strains isolated from raw chicken meat cuts samples . 2. MATERIAL AND METHODS 2.1.   Samples collection A grand total of 120 random samples (about 250 g for each) of fresh chicken meat cuts of Drumstick, thigh and breast (40 of each) were purchased from different supermarkets and retail chicken butchers at Kaliobia governorate. Each sample was kept in a separate sterile bag and transferred directly to lab without undue delay for bacteriological examination. 2.2.    Bacteriological examination 1. Preparation of samples (APHA, 2001) Twenty five grams of the sample were taken under aseptic condition to sterile Stomacher bag then 225 ml sterile 0.1%  peptone water were added. The contents were homogenized at Stomacher (MA 106402 France, 450 to 640 strokes per minute) for 2 minutes and the mixture was allowed to stand for 5 minutes at room temperature. The contents were transferred into sterile flask and thoroughly mixed by shaking and 1 ml was transferred into separate tube each containing 9 ml sterile 0.1% peptone water, from which tenth- fold serial dilutions were prepared. The  prepared samples were subjected to the following bacteriological examination: 2. Isolation and identification of suspected S. aureus (Quinn et al., 2002) 3. Detection of enterotoxins producing isolates by SET- RPLA technique (Igarashi et al., 1986). 4. Genotyping of enterotoxins virulence genes by using PCR (Sambrook et al., 1989). 3. RESULTS The results obtained in Table (2) revealed that, 33 isolates of coagulase positive S.aureus  were isolated from examined fresh chicken meat cuts (drumstick, thigh and breast samples represented as 13(32.5%) from drumstick samples; 11(27.5%) from thigh samples and 9(22.5%) from breast samples. Moreover, 87samples out of 120 ones were accepted, as they were free from coagulase Positive S.aureus  isolates according to EOS (2005). As shown in results of SET- RPLA test in table (3) 8 strains of 10 random examined strains (80.0%) were enterotoxigenic and classified according to type of toxin into 3A; 1C, 2D and 2A&C. The PCR results showed that,  sea  was detected in 4 studied strains (40.0%);  seb gene in one strain (10.0%) and  sec ;  sed   and  see  genes were detected in 3 strains (30.0% for each) as shown in Table (4). Regarding to enterotoxins A Fig., 1  Detection of virulence genes of enterotoxigenic Staphylococcus aureus   412 showed that, the  sea gene was amplified in 4 of studied S.aureus  strains (40.0%) at 102 bp. The  seb gene was amplified in one of studied S.aureus  strains (10.0%) at 164 bp (Fig. 2). Meanwhile, the  sec gene was amplified in 3 of studied S.aureus  strains (30.0%) at 451 bp (Fig. 3).In addition, the  sed  gene was amplified in 3 of studied S.aureus  strains (30.0%) at 278 bp (Fig. 4). Moreover, the results of PCR for amplification of  see gene in S.aureus  isolates (Fig. 5) showed that,  see gene was amplified in 3 of studied S.aureus  strains (30.0%) at 209 bp. Table (1): Oligonucleotide primers sequence, target genes, amplicons sizes and cycling conditions Table (2): Incidence of Coagulase Positive S. aureus  in examined samples of fresh chicken meat cuts (n=40 for each sample)  Samples No. Positive  No. of accepted samples  No. of non-accepted samples  No. %* Drumstick 40 13 32.5 27 13 Thigh 40 11 27.5 29 11 Breast 40 9 22.5 31 9 Total 120 33 27.5 87 33 * Percentage in relation to total number of sample in each row. Table (3): The incidence of enterotoxins production from isolated S.aureus  using SET-RPLA test   No. of S.aureus Enterotoxigenic strains Type of enterotoxin  NO. % A C D A&C 10 8 80.0 3 1 2 2   Hassanen et al. (2017)   .   BVMJ,   33,    N O .   2:   410-417   413 Table (4): The results of PCR amplifications of different used genes of S.aureus   S.aureus strains Virulence genes Sea Seb Sec Sed See 1 + - + - -2 - - - - +3 - - - - -4 + - + - -5 - - - + -6 - - - - +7 - - - - -8 - - + + -9 + + - - +10 + - - + -Total No.4 1 3 3 3% 40 10 30 30 30  sea (enterotoxin A)  seb (enterotoxin B),  sec (enterotoxin C)  sed  (enterotoxin D),  see ( enterotoxin E).   Fig (1): Agarose gel electrophoresis of enterotoxin A (  sea ) gene. Lane L: 100-600 bp DNA Ladder. Negative control (Listeria reference strain, NCINB 50007). Pos.: Positive control ( S.aureus  referencestrain at 102 bp). Lane 1; 4; 9&10: S.aureus  (Positive)at 102bp. Lane 1-8 and 10: S.aureus  (Negative) Fig (2): Agarose gel electrophoresis of enterotoxin A (  seb ) gene. Lane L: 100-600 bp DNA Ladder. Negative control (Listeria reference strain, NCINB 50007). Pos.: Positive control ( S.aureus  referencestrain at 164 bp). Lane 9: S.aureus  (Positive) at 102bp. Lane 1-8 and 10: S.aureus  (Negative) Fig (3): Agarose gel electrophoresis of enterotoxin A (  sec ) gene. Lane L: 100-600 bp DNA Ladder. Negative control (Listeria reference strain, NCINB 50007). Pos.: Positive control ( S.aureus  referencestrain at 451 bp). Lane 1; 4; 8: S.aureus  (Positive) at 451bp. Lane 2;3;5;6;7;9 &10:S.aure us  (Negative)   Fig (4): Agarose gel electrophoresis of enterotoxin A (  sed  ) gene. Lane L: 100-600 bp DNA Ladder. Negative control (Listeria reference strain, NCINB 50007). Pos.: Positive control ( S.aureus  reference strain at 278 bp). Lane 5; 8 &10: S.aureus (Positive) at 278bp. Lane 1;2;3;4;6;7 &9:S.aureus (Negative)  Detection of virulence genes of enterotoxigenic Staphylococcus aureus   414 Fig (5): Agarose gel electrophoresis of enterotoxin A (  see ) gene. Lane L: 100-600 bp DNA Ladder. Negative control (Listeria reference strain, NCINB 50007). Pos.: Positive control ( S.aureus  reference strain at 209 bp). Lane 2; 6 &9: S.aureus (Positive) at 209bp. Lane 1;3;4;5;7;8 &10:S.aureus (Negative)  4. DISCUSSION Chicken meat is a common vehicle of food  borne illness, Staphylococci mainly S. aureus  is the most important causes of food poisoning through enterotoxins (SEs)  production and the presence of these organisms has relevant public health implications (Prange et al., 2005 and Bhaisare et al., 2014) Most previous studies were interested with coagulase positive S.aureus  strains of Staphylococci species as it is still a major cause of food poisoning due to ingestion of enterotoxin (Le Loir, 2003) and the ability to produce such enterotoxin in food is more likely when competing microorganisms were absent, resulting in symptoms of intoxication and common symptoms appear approximately 3-8 hr. after ingestion as nausea, vomiting, abdominal cramps and diarrhea. Generally, symptoms are short in duration, “approximately 24 -48hrs (Sandle and Mckillip, 2004). The results obtained in table (2) came in accordance with those obtained by Enany et al .  (2013) they reported that 25% were  positive for S.aureus  Meanwhile: these results were disagreed with those of Mohamed- Ghada (2010) and Abo-Samra (2013). They cleared that; the incidence of S.aureus  in fresh chicken meat samples was 26 (52%). The presence of coagulase positive S. aureus  in chicken meat and its products indicates poor hygiene of meat handlers as well as lack of sterilization of utensils and they grow without pronounced change in odour or taste in the products and  producing heat stable enterotoxins which lead to food poisoning with severe diarrhoea and gastroenteritis among consumers (Le Loir, 2003andBakr  et al.,  2004). Staphylococcus auerus is important in relation to poultry meat hygiene because of its ability to produce enterotoxins. These enterotoxins are serologically grouped into four major classical types which are SEA, SEB, SEC and SED detected by SET-RPLA (Zouharova and Rysanek 2008) and Bendahou et al. (2009). This result nearly similar to that recorded by Afifi-Dina (2016) who mentioned that S.aureus  strains out of 10 randomly ones examined by SET- RPLA test were enterotoxigenic and classified according to type of toxin into 3A;1 B& 1C. The PCR results as shown in Table (4) regarding to enterotoxins A (Fig., 1) came in harmony with those of Nemati (2014); Abdalrahman et al. (2015) and Afifi-Dina (2016) who stated that enterotoxin A (  sea ) was detected in 2 studied strains only. Meanwhile, these results were disagreed with that of FeBler et al . (2011) who failed to detect  sea  gene in S.aureus  strains isolated from poultry and poultry products. According to Fig. 2 nearly similar results were obtained by Abdalrahman et al .  (2015) and Moustafa et al .  (2016) who found  seb (8.3%). Meanwhile according toFig., 3.nearly similar results were obtained by Chen et al. (2013); Madahi et al. (2014) who found that sec was 12.50% and Nemati (2014). In addition Fig4 nearly similar results were obtained by Pelisser et al. (2009); Madahi et al. (2014) 8.33%  sed and Moustafa et al. (2016). Moreover, the results of PCR in Fig., 5 were nearly similar to those obtained by Chiang et al. (2006) and Pelisser et al. (2009) who
Recommended
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x