Virulence Factors of Staphylococcus aureus Isolated from Korean Pork bulgogi: Enterotoxin Production and Antimicrobial Resistance.

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 mecA-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.

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 . However, a number of other enterotoxin types designated SEG-SEJ, SElM, SElN, and SElO has been defined (Argudín ; Yarwood ).

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 mecA and femA genes (Keun ). The mecA 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 ; Hong ; Jo ). Due to the high salt content of soy sauce, bulgogi does not provide an environment suitable for microbial growth (Jo ). As a result, just 5 strains were isolated from 90 pork bulgogi samples (Ahn ). 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 ). 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. aureus DNA 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 ; Yarwood ). 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.

Table 1.

Used oligonucleotide primers and amplification conditions

Genes	Primer	Oligonucleotide sequences	Product size (bp)	Annealing temp. (℃)
23S rRNA	STAUR2	ACGGAGTTACAAAGGACGAC	1,250	64
	STAUR4	AGCTCAGCCTTAACGAGTAC
nuc	nuc-1	GCGATTGATGGTGATACGGTT	279	55
	nuc-2	AGCCAAGCCTTGACGAACTAAAGC
sea	SEA-1	AAAGTCCCGATCAATTTATGGCTA	219	55
	SEA-2	GTAATTAACCGAAGGTTCTGTAGA
seb	SEB-1	TCGCATCAAACTGACAAACG	476	55
	SEB-2	GCAGGTACTCTATAAGTGCC
sec	SEC-1	GACATAAAAGCTAGGAATTT	257	55
	SEC-2	AAATCGGATTAACATTATCC
sed	SED-1	CTAGTTTGGTAATATCTCCT	317	55
	SED-2	TAATGCTATATCTTATAGGG
see	SEE-1	TAGATAAAGTTAAAACAAGC	169	55
	SEE-2	TAACTTACCGTGGACCCTTC
seg	SEG-1	AATTATGTGAATGCTCAACCCGATC	642	55
	SEG-2	AAACTTATATGGAACAAAAGGTACTAGTTC
seh	SEH-1	CAATCACATCATATGCGAAAGCAG	376	55
	SEH-2	CATCTACCCAAACATTAGCACC
sei	SEI-1	CTCAAGGTGATATTGGTGTAGG	577	55
	SEI-2	AAAAAACTTACAGGCAGTCCATCTC
sej	SEJ-1	CATCAGAACTGTTGTTCCGCTAG	192	55
	SEJ-2	CTGAATTTTACCATCAAAGGTAC
selm	SEM-1	TCTTAGGAACTATTATGGTAGC	471	55
	SEM-2	CCTGCATTAAATCCAGAA
seln	SEN-1	GGAGTTACGATACATGATGG	292	55
	SEN-2	ACTCTGCTCCCACTGAAC
selo	SEO-1	TGATGATTATATAAATAATCGATTTACG	249	55
	SEO-2	ATATGTACAGGCAGTATCC
mecA	mecA-1	TCACCTTGTCCGTAACCTGA	678	57
	mecA-2	TCGTGTCACAATCGTTGACG
femA	femA-1	CGAGGTCATTGCAGCTTGCT	231	57
	femA-2	CCAGCATTACCTGTAATCTCGCCA

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 of mecA and femA genes

The mecA and femA 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 the S. aureus strains isolated from pork bulgogi

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 ). In this study, the 4 isolates were confirmed as S. aureus strains by PCR amplification of the 23S rRNA and nuc genes (Table 2).

Table 2.

Characteristics of isolated S. aureus strains from pork bulgogi

Isolate	23S rRNA gene	nuc gene	SE genes	femA gene	mecA gene
S10-2	+	+	seg, seh, sei, sej, selm, seln	+	+
S10-3	+	+	sea, seh	+	−
S12-2	+	+	sea, seh	+	−
S13-2	+	+	sea, sed, seh	+	−

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 ). 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 ; Zschck ). In addition, SEG, SEH, and SEI have also been shown to be emetic after oral administration in a primate model (Argudín ).

Fig. 1.

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.

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 , who reported that ampicillin and penicillin resistance was frequently founded at a ratio of 70 and 73%, respectively.

Table 3.

Antibiotic resistance of control strains and isolated S. aureus from pork bulgogi

Antibiotic groups	Antibiotic	Strains
		KCCM 11593	ATCC 25923	KCCM 40510	KCCM 40511	KCCM 40512	S10-2	S10-3	S12-2	S13-2
Tetracyclines	Tetracycline (30 μg)	Sa	S	Rb	S	S	R	R	R	R
Aminoglycosides	Streptomycin (10 μg)	S	S	R	S	S	S	S	S	S
	Gentamicin (30 μg)	S	S	S	R	S	S	S	S	S
Glycopeptide	Vancomycin (30 μg)	S	S	S	S	S	S	S	S	S
Amino penicillin	Amoxicillin (20 μg)+Clavulanic acid (10 μg)	S	S	R	S	S	S	S	S	S
β-Lactam	Ampicillin (10 μg)	S	S	R	R	R	R	S	S	R
	Penicillin (10 IU)	S	S	R	R	R	R	S	S	R
	Oxacillin (10 μg)	S	S	R	R	R	R	S	S	S
Cephalosporins	Cefazoline (30 μg)	S	S	R	S	S	S	S	S	S
	Cephalothin (30 μg)	S	S	R	S	S	S	S	S	S

aSensitive; bresistant.

Table 2 shows results for detection of mecA and femA genes in the S. aureus strains isolated from pork bulgogi. All the tested strains possessed the femA gene. The MRSA strains KCCM 40510 and KCCM 40511 were positive for the mecA gene. Although the mecA 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 mecA gene; consequently, this strain was identified as a MRSA. Although the mecA 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 mecA 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 ).

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.