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Virulence Factors of Staphylococcus aureus Isolated from Korean Pork bulgogi: Enterotoxin Production and Antimicrobial Resistance
Virulence Factors of Staphylococcus aureus Isolated from Korean Pork bulgogi: Enterotoxin Production and Antimicrobial Resistance
Food Science of Animal Resources. 2015. Aug, 35(4): 502-506
Copyright © 2015, Korean Society for Food Science of Animal Resources
This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licences/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : June 15, 2015
  • Accepted : July 20, 2015
  • Published : August 31, 2015
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About the Authors
Byeong Su, Jung
Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 143-701, Korea
Yong Ju, Lee
Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 143-701, Korea
Na-Kyoung, Lee
Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 143-701, Korea
Hyoun Wook, Kim
Animal Products and Development division, National Institute of Animal Science, RDA, Suwon 441-706, Korea
Mi-Hwa, Oh
Animal Products and Development division, National Institute of Animal Science, RDA, Suwon 441-706, Korea
Hyun-Dong, Paik
Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea
hdpaik@konkuk.ac.kr

Abstract
The aim of this study was to investigate the antimicrobial resistance profiles of and the enterotoxin gene distribution in 4 strains of Staphylococcus aureus (S10-2, S10-3, S12-2, and S13-2) isolated from 90 bulgogi samples. The S. aureus enterotoxin H gene ( seh ) was found in all the strains, while the S. aureus enterotoxin A gene ( sea ) was found only in 3 of the 4 strains. The S10-2 strain expressed a combination of enterotoxin genes - seg, seh, sei, sej, selm , and seln . The strains S10-2 and S13-2 were resistant to ampicillin and penicillin G, and all the isolated strains were resistant to tetracycline. The S10-2 strain was the only mec A-positive strain; it was also resistant to β-lactam antibiotics. Thus, genes encoding enterotoxin as well as those conferring antibiotic resistance were identified in the S. aureus strains isolated from pork bulgogi . These results represents the potential occurrence of MRSA in pork bulgogi , and the need for a monitoring system for pork bulgogi in order to prevent an outbreak of staphylococcal food poisoning.
Keywords
Introduction
Staphylococcus aureus is one of the most common causes of food borne diseases and is found in a variety of foods (Andreja, 2012). S. aureus is a public health concern because of its ability to produce enterotoxins and to survive in harsh conditions. Staphylococcal enterotoxins (SEs) are a leading cause of staphylococcal food poisoning in humans; they may also be involved in other types of infections. The symptoms of SE poisoning are increased saliva secretion, vomiting, abdominal cramping, and diarrhea, with blood in some cases. Staphylococcal enterotoxins A (SEA) through to staphylococcal enterotoxin E (SEE) are the most commonly isolated enterotoxins in food poisoning outbreaks (Argudín ., 2010) . However, a number of other enterotoxin types designated SEG-SEJ, SE l M, SE l N, and SE l O has been defined ( Argudín ., 2010 ; Yarwood ., 2002 ).
The extensive therapeutic use of antibiotics in humans and animals has contributed to the increase in antibiotic resistance in pathogens. Especially problematic among these resistant pathogens are methicillin-resistant S. aureus (MRSA), which have become a serious concern due to the high rates of community and nosocomial-acquired infections. In addition, the phenomenon of horizontal gene transfer has resulted in the spread of methicillin resistance, increasing the pressure on a limited supply of alternative antibiotics. Antibiotic resistance in MRSA is determined by the mec A and fem A genes ( Keun ., 2011 ). The mec A gene encodes a penicillin binding protein (PBP-2a), which has a low binding affinity for methicillin and other β-lactam antibiotics.
Pork bulgogi is barbecued or pan-fired pork that has been marinated with a mixture of soy sauce, sugar, minced garlic, green onion, black pepper, and sesame oil. Its microbial safety was reported their bacterial condition ( (Ahn ., 2012 ; Hong ., 2011 ; Jo ., 2004 ). Due to the high salt content of soy sauce, bulgogi does not provide an environment suitable for microbial growth ( Jo , 2004 ). As a result, just 5 strains were isolated from 90 pork bulgogi samples ( Ahn ., 2012 ). The low numbers of S. aureus isolates may be due to soy sauce, which has been shown to exert antimicrobial effects against S. aureus , Shigella flexneri , Vibrio cholera , Salmonella Enteritidis, and Escherichia coli ( Kataoka, 2005 ). The aim of this study was to investigate the antimicrobial resistance profiles of and the enterotoxin gene distribution in S. aureus strains isolated from pork bulgogi .
Materials and Methods
- Bacterial strains
The four S. aureus strains S10-2, S10-3, S12-2, and S13-2 were isolated from raw pork bulgogi using Baird-Parker agar plates enriched with egg yolk (EY) tellurite ( Ahn ., 2012 ). Five different S. aureus strains KCCM 11593, ATCC 25923, KCCM 40510, KCCM 40511, and KCCM 40512 were used as standards. Frozen stocks were maintained at −80℃ in tryptic soy broth (TSB, Difco Laboratories, USA) containing 20% glycerol. The S. aureus strains were incubated at 37℃ for 24 h in TSB, and used for further study.
- S. aureusDNA extraction
Following overnight growth, the S. aureus strains were harvested by centrifugation at 8,900 × g for 15 min. The pelleted cells were used for genomic DNA extraction using the AccuPrep ® genomic DNA extraction kit (Bioneer Co., Korea), together with lysozyme treatment (Sigma Chemical Co., USA), according to the manufacturer’s instructions.
- PCR detection of Staphylococcal enterotoxin genes
PCR was used to test for the presence of 23S rRNA, nuc , and staphylococcal enterotoxin (SE) genes defined ( Argudín ., 2010 ; Yarwood ., 2002 ). The PCR reaction was performed in a 20 μL reaction volume containing 10 pmol of each primers ( Table 1 ), 25 μL of 2× Go Taq ® Green Master Mix (Promega, USA), 2 μL of template DNA. The final volume was adjusted to 50 μL using RNase free sterile water. All amplification steps were carried out in a thermocycler (Bioer, Switzerland) with an initial denaturation step at 94℃ for 5 min, followed by 30 cycles of denaturation at 94℃ for 2 min, annealing at 55-64℃ for 30 s, and a 30 s extension at 72℃, follow by a final extension at 72℃ for 10 min. The amplified PCR products were resolved by electrophoresis in a 1% agarose gel.
Used oligonucleotide primers and amplification conditions
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Used oligonucleotide primers and amplification conditions
- Antibiotic susceptibility
Antibiotic susceptibility of the S. aureus strains were tested using the disc agar diffusion method on Mueller Hinton agar (Dection Dickinson, France), following the guidelines of the Clinical and Laboratory Standards Institute ( CLSI, 2007 ). The antibiotic discs used were as tetracycline (30 μg), streptomycin (10 μg), gentamicin (30 μg), vancomycin (30 μg), amoxicillin (20 μg) and clavulanic acid (10 μg), ampicillin (10 μg), penicillin (10 IU), oxacillin (10 μg), cefazolin (30 μg), and cephalothin (30 μg). The strains were classified as susceptible, intermediate, or resistant according to the supplier’s instructions.
- Detection ofmecA andfemA genes
The mec A and fem A genes primers used as a biomarker for resistance. The FastPCR software program (http://primerdigital.com/fastpcr.hml) was used to analyze primers ( Table 1 ) for self-dimerization, cross-dimerization, and optimum annealing temperature. PCR conditions was followed manufacture’s instruction, and annealing temperatures are shown in Table 1 .
Results and Discussion
- Detection of enterotoxin genes of theS. aureusstrains isolated from porkbulgogi
The S. aureus strains were isolated from pork bulgogi using a selective medium in our previous study and were identified as S. aureus by Gram staining, catalase testing, latex agglutination, and the API Staph System ( Ahn ., 2012 ). In this study, the 4 isolates were confirmed as S. aureus strains by PCR amplification of the 23S rRNA and nuc genes ( Table 2 ).
Characteristics of isolatedS. aureusstrains from porkbulgogi
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Characteristics of isolated S. aureus strains from pork bulgogi
Staphylococcal enterotoxins (SEs) belong to a large family of staphylococcal pyrogenic exotoxins, which have been implicated in food poisoning, causing toxic shock-like syndromes, and several allergic and autoimmune diseases ( Ortega ., 2010 ). Fig. 1 shows the results for the PCR detection of the enterotoxin genes sea to selo . The strain S10-2 possessed 6 enterotoxin genes: seg , seh , sei , sej , selm , and seln . Strains S10-3 and S12-2 possessed the sea and seh enterotoxin genes. The strain S13-2 was shown to possess 3 genes encoding SEA, SED, and SEH. The seh gene, which encodes SEH, was detected in all the strains; however, there was no amplification of the enterotoxin genes seb , sec , see , and selo . Results of enterotoxin genes had a different as the strains. The enterotoxins SEA, SEB, and SED are the most common enterotoxins associated with human food-poisoning outbreaks ( Argudín ., 2010 ; Zschck ., 2005 ). In addition, SEG, SEH, and SEI have also been shown to be emetic after oral administration in a primate model ( Argudín ., 2010 ).
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Agarose gel electrophoresis analysis of PCR products of enterotoxin genes. (A) 10-2, (B) 10-3, (C) 12-2, (D) 13-2. Lane 1, sea; lane 2, seb; lane 3, sec; lane 4, sed; lane 5, see; lane 6, seg; lane 7, seh; lane 8, sei; lane 9, sej; lane 10, selm; lane 11, seln; lane 12, selo.
- Antibiotic susceptibility
S. aureus infections are primarily controlled through antibiotics. It is therefore concerning how rapidly this organism develops resistance to a large number of antibiotics. Here, we compared the antibiotic resistance of the S. aureus strains isolated from pork bulgogi to that of the standard strains (KCCM 11593 and ATCC 25923) and the MRSA strains (KCCM 40510, KCCM 40511, and KCCM 40512) ( Table 3 ). As expected the standard S. aureus strains demonstrated no antibiotic resistance. The KCCM 40510 strain was resistant to tetracycline, streptomycin, amoxicillin and clavulanic acid, ampicillin, penicillin, oxacillin, cefazoline, and cephalothin. The strains KCCM 40511 and KCCM 40512 were resistant to ampicillin, penicillin G, and oxacillin. The S. aureus isolates S10-2, S10-3, S12-2, and S13-2 strains were all resistant to tetracycline, whereas the S10-2 and S13-2 strains were resistant to ampicillin and penicillin G. Thus, the resistance profile of the isolated strains was similar to that of the MRSA strains, KCCM 40511 and KCCM 40512. Our findings are also consistent with those of Pereira . (2009) , who reported that ampicillin and penicillin resistance was frequently founded at a ratio of 70 and 73%, respectively.
Antibiotic resistance of control strains and isolatedS. aureusfrom porkbulgogi
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aSensitive; bresistant.
- Detection ofmecA andfemA genes
Table 2 shows results for detection of mec A and fem A genes in the S. aureus strains isolated from pork bulgogi . All the tested strains possessed the fem A gene. The MRSA strains KCCM 40510 and KCCM 40511 were positive for the mec A gene. Although the mec A gene was not detected in the S. aureus strain KCCM 40512, this strain was resistant to oxacillin. Among the strains isolated from pork bulgogi , only the strain S10-2 possessed the mec A gene; consequently, this strain was identified as a MRSA. Although the mec A gene was not detected in the S13-2 strain, antibiotic susceptibility testing revealed resistance to the β-lactam antibiotics, ampicillin, and penicillin. This may be due to a modified mec A gene, which was not detected by the primers used in this study. Antibiotic resistance can also be plasmid encoded rather than chromosomal ( Lee and Lim, 2002 ). The MRSA strains isolated from pork bulgogi were resistant to a higher number of antibiotics than the methicillin-sensitive strains were. This is consistent with the finding that MRSA strains generally express genes that confer resistance to multiple antibiotics ( Kumar ., 2011 ).
The S10-2 and S13-2 strains were both resistant to ampicillin and penicillin G. However, the S10-2 stain expressed 6 enterotoxin genes ( seg , seh , sei , sej , selm , and seln ), while the S13-2 strain possessed only 3 enterotoxin genes ( sea , sed , and seh ). These results represents the potential occurrence of MRSA in pork bulgogi , and the need for a monitoring system for pork bulgogi in order to prevent an outbreak of staphylococcal food poisoning.
Acknowledgements
This work was supported by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093824).
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