Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus Isolates from Louisiana Retail Meats

Abstract

Staphylococcus aureus is a leading cause of food poisoning worldwide due to the production of heat-stable enterotoxins. Recently, the isolation of methicillin-resistant S. aureus (MRSA) from food animals and retail meats raised additional food safety concerns. In this study, we characterized 152 S. aureus isolates, including 22 MRSA recovered from Louisiana retail pork and beef meats, for the prevalence of nine enterotoxin and four other exotoxin genes by polymerase chain reaction and antimicrobial susceptibility testing by broth microdilution. Overall, 85% of S. aureus isolates were positive for at least one of six enterotoxin genes identified and 66% harbored two to four enterotoxin genes. The two most predominant ones were seg and sei (66% each), followed by seh (20%), sed (15%), sej (13%), and sea (1%). No isolates harbored enterotoxin genes seb, sec, or see, the toxic shock syndrome toxin 1 gene tst, or the exfoliative toxin genes eta or etb. Three MRSA isolates were the only ones harboring Panton-Valentine leucocidin. Resistances were common to penicillin (71%), ampicillin (68%), and tetracycline (67%), followed by erythromycin (30%), clindamycin (18%), oxacillin with 2% NaCl (14%), ciprofloxacin (13%), levofloxacin (13%), gentamicin (3%), quinupristin/dapfopristin (3%), chloramphenicol (2%), and moxifloxacin (1%). Multidrug resistance was commonly observed among MRSA isolates and S. aureus isolates from pork. This study demonstrated that S. aureus isolates found in Louisiana retail pork and beef meats possessed various enterotoxin genes and antimicrobial resistance profiles. Therefore, vigilant food safety practice needs to be implemented for people who handle raw meat products to prevent foodborne infections and intoxications due to S. aureus contamination.

Introduction

S taphylococcus aureus is a major cause of food poisoning worldwide due to heat-stable staphylococcal enterotoxins (SEs) produced by enterotoxigenic strains (Le Loir et al., 2003). Ingestion of preformed SEs in foods can rapidly lead to staphylococcal food poisoning (SFP) with symptoms such as vomiting, nausea, abdominal cramps, and diarrhea (Seo and Bohach, 2007). In the United States, SFP is estimated to account for 185,060 illnesses and 1753 hospitalizations annually (Mead et al., 1999). Since most SFP cases are mild, the actual number of SFP is estimated to be much higher than those reported (Mead et al., 1999).

SEs belong to a large pyrogenic toxin family (Dinges et al., 2000). Besides functioning as potent emetic agents, SEs also act as superantigens that stimulate nonspecific T-cell proliferation and cause toxic shock (Balaban and Rasooly, 2000). Twenty-one serologically distinct SE or SE-like toxins have been identified to date, which include the classical SEs (SEA through SEE), newer SEs (SEG through SEJ), and more recent ones (SEK through SEV) (Thomas et al., 2006, 2007; Ono et al., 2008). Some newly described SEs lack emetic activity and their involvement in SFP is not clear (Balaban and Rasooly, 2000). Previously, SEA and SED were reported to be the two predominant SEs identified in cases of SFP, followed by SEB (Balaban and Rasooly, 2000). However, such trend appears to change with time (Seo and Bohach, 2007). To detect these toxins, immunological assays such as immunodiffusion, agglutination, and enzyme-linked immunosorbent assay have been widely used; however, those assays are limited to detecting classical SEs only (Su and Lee Wong, 1997). Recently, polymerase chain reaction (PCR) assays have been developed to detect the prevalence of many SE genes and were reported to be efficient and reliable (Monday and Bohach, 1999; McLauchlin et al., 2000; Mehrotra et al., 2000).

Besides SEs, S. aureus is capable of producing an array of other exotoxins, which serve as important virulence factors for causing nosocomial and community-associated invasive S. aureus infections. These exotoxins include the toxic shock syndrome toxin 1 (TSST-1, formerly SEF), exfoliative toxins A and B (ETA and ETB), and Panton-Valentine leucocidin (PVL) (Dinges et al., 2000). TSST-1 is also a pyrogenic toxin superantigen and it is the first toxin shown to be involved in toxic shock syndrome (Schlievert et al., 1981). The two ETs, in conjunction or alone, are implicated in staphylococcal scalded-skin syndrome (Iandolo, 1989). PVL is a two-component staphylococcal membrane toxin that targets leukocytes. It is widely distributed among some community-associated methicillin-resistant S. aureus (CA-MRSA) clones and may contribute to the increased virulence of CA-MRSA strains (Lina et al., 1999).

In recent years, the emerging antimicrobial resistance occurring in S. aureus, particularly MRSA in both nosocomial and community settings, has become a major public health concern (Kennedy et al., 2008). Very recently, multiple studies have demonstrated the presence of MRSA in food animals, people in contact with these animals, and retail meats (de Neeling et al., 2007; van Loo et al., 2007; Wulf et al., 2008; Smith et al., 2009). In a survey of pork and beef products in Louisiana retail stores, we reported that 47 (39.2%) out of 120 samples contained S. aureus, 6 of which were positive for MRSA (Pu et al., 2009). These studies raised additional food safety concerns of S. aureus beyond an agent causing food poisoning (Kluytmans, 2010).

Previous food safety research on S. aureus focused on the characterization of SE productions; however, relative little is known about the antimicrobial susceptibility profiles of enterotoxigenic S. aureus strains or the prevalence of SE and other virulence factors among MRSA isolates. The objective of this study was to characterize both the prevalence of enterotoxin and other exotoxin genes and the antimicrobial susceptibility profiles of 152 S. aureus isolates, including 22 MRSA from Louisiana retail meat.

Materials and Methods

Bacterial strains and culture conditions

The 152 S. aureus isolates used in this study (Table 1) were recovered previously from 56 retail pork and beef samples obtained from five supermarket chains in Baton Rouge, Louisiana (Pu et al., 2009). For the majority (38 out of 56) of S. aureus–positive meat samples, three isolates were included. The procedure used to recover S. aureus from raw meats included enrichment in trypticase soy broth (BD Diagnostic Systems, Sparks, MD) supplemented with 10% NaCl and 1% sodium pyruvate (Sigma-Aldrich, St. Louis, MO), followed by selective plating on Baird-Parker medium (BD Diagnostic Systems) with or without 4 μg/mL of cefoxitin (Pu et al., 2009). Twenty-two out of the 152 S. aureus isolates were confirmed to be MRSA based on specific amplification of the mecA gene, among which 19 were recovered from one beef and three pork samples in a single grocery store of Chain A and determined to be MRSA clone USA100, whereas the remaining 3 were recovered from two pork samples in two grocery stores of Chain B and were identified to be MRSA clone USA300 (Pu et al., 2009). Additionally, the three USA300 isolates were found to be PVL positive (Pu et al., 2009). The cultures were stored in trypticase soy broth containing 20% glycerol at −80°C, and grown routinely on trypticase soy agar (TSA; BD Diagnostic Systems) and incubated at 35°C for 24 h.

Table 1.

The Source and Origin of 152 Staphylococcus aureus Isolates Used in This Study

Supermarket chain	Meat type	No. (%) of Staphylococcus aureus isolates recovered from	No. (%) of S. aureus–positive meat samples recovered from
A	Pork	26 (17)	8 (14)
	Beef	10 (7)	3 (5)
	Subtotal	37 (24)	11 (20)
B	Pork	55 (36)	21 (38)
C	Pork	17 (11)	8 (14)
	Beef	1 (1)	1 (2)
	Subtotal	18 (12)	9 (16)
D	Pork	9 (6)	3 (5)
	Beef	3 (2)	1 (2)
	Subtotal	12 (8)	4 (7)
E	Pork	28 (18)	10 (18)
	Beef	3 (2)	1 (2)
	Subtotal	31 (20)	11 (20)
All	Pork	135 (89)	50 (89)
	Beef	17 (11)	6 (11)
	Total	152 (100)	56 (100)

Detection of toxin genes

To prepare DNA templates for PCR, a single S. aureus colony grown on TSA plates was suspended in 0.5 mL of TE buffer (10 mM Tris, pH 8.0; 1 mM ethylenediaminetetraacetic acid) and heated at 95°C for 10 min in a dry heating block. The crude cell lysate was centrifuged at 12,000 g for 2 min and the supernatant was stored at −30°C until use.

Table 2 lists the primer sets used to detect nine SE genes (sea, seb, sec, sed, see, seg, seh, sei, and sej), the TSST-1 gene (tst), the ETA and ETB genes (eta and etb), and the PVL genes (lukS and lukF). Two sets of multiplex PCR were performed as indicated in Table 2 and a single PCR was conducted for the PVL genes. Each PCR mix in a total volume of 25 μL consisted of the following: 1 × PCR buffer, 0.2 mM of each deoxynucleotide triphosphate, 2.5 mM of MgCl₂, 0.625 unit of Go Taq Hot Start Polymerase (Promega, Madison, WI), 0.3 μM of each primer (Invitrogen, Carlsbad, CA), and 5 μL of DNA template. The PCR was conducted using 95°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing at 57°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 7 min in a Bio-Rad C1000 Thermal Cycler (Hercules, CA). PCR products were analyzed by electrophoresis on 1.5% agarose gel containing ethidium bromide, observed under UV light, and documented with a Gel Doc XR system (Bio-Rad).

Table 2.

Primers Used to Characterize Staphylococcal Enterotoxin and Other Exotoxin Genes

Target gene	Primer sequences (5′–3′)	Amplicon size (bp) ^a	Multiplex polymerase chain reaction set	Reference
sea	GCA GGG AAC AGC TTT AGG C	521	A	Monday and Bohach (1999)
	GTT CTG TAG AAG TAT GAA ACA CG
seb-sec	ATG TAA TTT TGA TAT TCG CAG TG	665	A
	TGC AGG CAT CAT ATC ATA CCA
sec	CTT GTA TGT ATG GAG GAA TAA CAA	284	A
	TGC AGG CAT CAT ATC ATA CCA
sed	GTG GTG AAA TAG ATA GGA CTG C	385	A
	ATA TGA AGG TGC TCT GTG G
see	TAC CAA TTA ACT TGT GGA TAG AC	171	A
	CTC TTT GCA CCT TAC CGC
seg	CGT CTC CAC CTG TTG AAG G	328	A
	CCA AGT GAT TGT CTA TTG TCG
seh	CAA CTG CTG ATT TAG CTC AG	359	B
	GTC GAA TGA GTA ATC TCT AGG
sei	CAA CTC GAA TTT TCA ACA GGT AC	466	B
	CAG GCA GTC CAT CTC CTG
sej	CAT CAG AAC TGT TGT TCC GCT AG	142	B
	CTG AAT TTT ACC ATC AAA GGT AC
tst	GCT TGC GAC AAC TGC TAC AG	560	B
	TGG ATC CGT CAT TCA TTG TTA A
eta	GCA GGT GTT GAT TTA GCA TT	93	B	Mehrotra et al. (2000)
	AGA TGT CCC TAT TTT TGC TG
etb	ACA AGC AAA AGA ATA CAG CG	226	B
	GTT TTT GGC TGC TTC TCT TG
lukS-lukF	ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A	433	Single polymerase chain reaction	Lina et al. (1999)
	GCA TCA AST GTA TTG GAT AGC AAA AGC

Differences in amplicon size were noted from those originally published after sequence analysis.

Antimicrobial susceptibility testing

Minimal inhibitory concentrations (MICs) for S. aureus isolates were determined by broth microdilution using Sensititre GPALL1F Gram Positive MIC Plates (Trek Diagnostic Systems, Cleveland, OH), following guidelines of the manufacturer and the Clinical and Laboratory Standards Institute (CLSI, 2006a). The GPALL1F Gram Positive MIC Plate contains U.S. Food and Drug Administration–cleared broth D-Test, along with Cefoxitin Screen and Daptomycin to eliminate offline testing. S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used as the quality control organisms. Briefly, three to five S. aureus colonies grown on fresh TSA plates were emulsified in sterile water and adjusted to a 0.5 McFarland Standard. Ten μL of the suspension was transferred into a tube containing 10 mL of cation-adjusted Mueller-Hinton broth, and 50 μL of the broth suspension was inoculated into the wells of the MIC plate. The plates were read after incubation at 35°C for 18–24 h. MICs were interpreted according to the guidelines of the manufacturer and CLSI (2006b). Antimicrobials in the panel and their resistance breakpoints were as following: ampicillin (≥0.5 μg/mL), cefoxitin screen (growth), chloramphenicol (≥32 μg/mL), ciprofloxacin (≥4 μg/mL), clindamycin (≥4 μg/mL), D test (growth), daptomycin (>1 μg/mL), erythromycin (≥8 μg/mL), gentamicin (≥16 μg/mL), levofloxacin (≥8 μg/mL), linezolid (>4 μg/mL), moxifloxacin (≥8 μg/mL), nitrofurantoin (≥128 μg/mL), oxacillin +2% NaCl (≥4 μg/mL), penicillin (≥0.25 μg/mL), quinupristin/dalfopristin (≥4 μg/mL), rifampin (≥4 μg/mL), tetracycline (≥16 μg/mL), tigecycline (>0.5 μg/mL), trimethoprim/sulfamethoxazole (≥4/76 μg/mL), and vancomycin (≥32 μg/mL).

Statistical analysis

The prevalence of each SE gene profile among S. aureus isolates between beef and pork was compared by using the Chi-square (χ ²) test (SAS for Windows, version 9; SAS Institute, Cary, NC). Differences between the prevalence rates were considered significant when p < 0.05.

Results

Prevalence of SE genes

Among 152 S. aureus isolates, 129 (85%) showed PCR amplifications of at least one of six SE genes identified, sea, sed, seg, seh, sei, and sej (Table 3). The two most predominant SE genes were seg and sei (100/152; 66% each), followed by seh (30/152; 20%), sed (23/152; 15%), sej (19/152; 13%), and sea (1/152; 1%). No isolates harbored three other SE genes, that is, seb, sec, or see. The overall prevalence of SE genes among pork isolates (117/135; 87%) was higher than that among beef isolates (12/17; 71%; Table 3). Of 56 S. aureus–positive meat samples, 51 (91%) contained S. aureus isolates harboring SE genes. Similarly, a higher percentage of pork samples (46/50; 92%) contained enterotoxigenic S. aureus than that of beef samples (5/6; 83%; Table 3). Nonetheless, the differences in the prevalence of enterotoxigenic S. aureus isolates observed between pork and beef were not statistically significant (p > 0.05). Noticeably, the only meat sample containing sea-positive S. aureus was a pork sample.

Table 3.

Prevalence of Staphylococcal Enterotoxin Gene Profiles Among 152 Staphylococcus aureus Isolates Recovered from Retail Beef and Pork Samples

	No. (%) of S. aureus isolates possessing a specific profile ¹			No. (%) of meat samples with a specific profile ²
SE gene profile	Beef (n = 17)	Pork (n = 135)	Subtotal (n = 152)	Beef (n = 6)	Pork (n = 50)	Subtotal ³ (n = 56)
sed-seg-seh-sei		1 (1)	1 (1)		1 (2)	1 (2)
sed-seg-sei-sej	5 (29)^A	14 (10)^B	19 (13)	1 (17)^a	3 (6)^a	4 (7)
sea-seg-sei		1 (1)	1 (1)		1 (2)	1 (2)
sed-seg-sei		3 (2)	3 (2)		1 (2)	1 (2)
seg-sei	3 (18)^B	73 (54)^A	76 (50)	2 (33)^a	31 (62)^a	33 (59)
seh	4 (24)^A	25 (19)^A	29 (19)	2 (33)^a	12 (24)^a	14 (25)
Any SE gene	12 (71)^A	117 (87)^A	129 (85)	5 (83)^a	46 (92)^a	51 (91)
None	5 (29)^A	18 (13)^A	23 (15)	2 (33)^a	8 (16)^a	10 (18)

In each row, percentages followed by different upper case letters are significantly different (p < 0.05) in terms of the prevalence of that specific SE gene profile between beef and pork isolates.

In each row, percentages followed by same lower case letters are not significantly different (p < 0.05) in terms of the prevalence of that specific SE gene profiles between S. aureus–positive beef and pork samples.

Certain meat samples (one beef and four pork) contained S. aureus isolates with two SE gene profiles and two pork samples contained S. aureus isolates with three SE gene profiles.

SE, staphylococcal enterotoxin.

For the five supermarket chains (A through E) where S. aureus isolates were recovered, the prevalence of enterotoxigenic S. aureus isolates ranged from 61% in chain C to 100% in chain A, whereas the prevalence rates of meat samples containing enterotoxigenic S. aureus isolates ranged from 78% in chain C to 100% in both chains A and D (data not shown).

Table 3 lists the SE gene profiles among the 152 S. aureus isolates recovered from 56 meat samples. Overall, the most commonly identified SE gene profile was the seg-sei combination, occurring in 50% of S. aureus isolates or 59% of S. aureus–positive meat samples. There were 19% of S. aureus isolates (25% of meat samples) harbored the seh gene only, whereas 13% of isolates (7% of meat samples) possessed the sed-seg-sei-sej four gene combination. One hundred (66%) S. aureus isolates (70% of meat samples) harbored two to four SE genes. Noticeably, all 100 seg-positive S. aureus isolates were positive for sei. Additionally, all 23 isolates harboring sed were positive for seg-sei and all 19 isolates harboring sej had the sed-seg-sei-sej profile (Table 3). The majority (85%; 41/48) of meat samples containing multiple S. aureus isolates had the same SE gene profiles for all isolates within the same sample (data not shown).

Among MRSA isolates, the 19 USA100 isolates possessed the same SE gene profile, sed-seg-sei-sej, which was unique to this group. Of the three USA300 MRSA isolates, one harbored seh only and the other two were negative for all nine SE genes tested.

Prevalence of other exotoxin genes

None of the 152 S. aureus isolates harbored the TSST-1 gene (tst) or the ET genes (eta or etb), and the only S. aureus isolates tested positive for the PVL genes were the three USA300 isolates.

Antibiotic susceptibility profiles

Table 4 shows the antimicrobial susceptibility testing results. All of the 152 S. aureus isolates were susceptible to daptomycin, linezolid, nitrofurantoin, rifampin, tigecycline, trimethoprim/sulfamethoxazole, and vancomycin. Resistances were common to penicillin (108/152; 71%), ampicillin (104/152; 68%), and tetracycline (102/152; 67%), followed by erythromycin (46/152; 30%), clindamycin (27/152; 18%), oxacillin with 2% NaCl (22/152; 14%), ciprofloxacin (19/152; 13%), levofloxacin (19/142; 13%), gentamicin (5/152; 3%), quinupristin/dapfopristin (4/152; 3%), chloramphenicol (3/152; 2%), and moxifloxacin (1/152; 1%). The 22 MRSA were the only ones positive for the cefoxitin screen, suggesting oxacillin resistance, which matched with the oxacillin with 2% NaCl result. Discordance in erythromycin and clindamycin resistance was observed in 19 S. aureus isolates, all being MRSA USA100 and demonstrating erythromycin-resistant and clindamycin-susceptible phenotype. All 19 isolates were positive for the D test, suggesting inducible clindamycin resistance.

Table 4.

Antimicrobial Susceptibility Among All 152 Staphylococcus aureus Isolates (22 Methicillin-Resistant Staphylococcus aureus and 130 Methicillin-Susceptible Ones) ^a

		Among all S. aureus isolates (n = 152)			Among MRSA isolates (n = 22)			Among other S. aureus isolates (n = 130)
Antimicrobial		No. of isolates in category			No. of isolates in category			No. of isolates in category
Subclass	Agent	S	I	R	S	I	R	S	I	R
Aminoglycosides	Gentamicin	147	0	5	19	0	3	128	0	2
Cephems	Cefoxitin screen^b	130 (−)	N/A	22 (+)	0 (−)	N/A	22 (+)	130 (−)	N/A	0 (+)
Fluoroquinolones	Ciprofloxacin	131	2	19	3	0	19	128	2	0
	Levofloxacin	133	0	19	3	0	19	130	0	0
	Moxifloxacin	146	5	1	16	5	1	130	0	0
Lincosamides	Clindamycin^c	124	1	27	19	0	3	105	1	24
Macrolides	Erythromycin	96	10	46	0	0	22	96	10	24
Penicillins	Ampicillin	48	0	104	0	0	22	48	0	82
	Oxacillin + 2%NaCl	130	0	22	0	0	22	130	0	0
	Penicillin	44	0	108	0	0	22	44	0	86
Phenicols	Chloramphenicol	44	105	3	4	18	0	40	87	3
Streptogramin	Quinupristin/dalfopristin	142	6	4	22	0	0	120	6	4
Tetracyclines	Tetracycline	50	0	102	19	0	3	31	0	99

All of the 152 S. aureus isolates were susceptible to daptomycin, linezolid, nitrofurantoin, rifampin, tigecycline, trimethoprim/sulfamethoxazole, and vancomycin.

For cefoxitin screen, positive and negative are indicated instead of S/I/R.

For clindamycin, 19 MRSA USA100 isolates were inducible clindamycin resistant.

MRSA, methicillin-resistant S. aureus; S, susceptible; I, intermediate; R, resistant; N/A, not applicable.

As indicated in Table 4, all of the 22 MRSA isolates were resistant to macrolides (erythromycin) and penicillins (ampicillin, oxicillin +2% NaCl, and penicillin), and were positive for the cefoxitin screen. In addition, the 19 USA100 isolates were resistant to two fluoroquinolones (ciprofloxacin and levofloxacin) and 1 USA100 isolate was resistant to moxifloxacin and 5 were in the intermediate category for this fluoroquinolone. All of the 19 USA100 isolates showed inducible resistance to a lincosamides, clindamycin. For the three USA300 isolates, besides macrolides and penicillins, resistances to aminoglycosides (gentamicin), lincosamides (clindamycin), and tetracyclines (tetracycline) were observed (Table 4).

Among the 130 methicillin-susceptible S. aureus isolates, resistances were common to tetracycline (99/130; 76%), penicillin (86/130; 66%), and ampicillin (82/130; 63%; Table 4). All 24 (18%) methicillin-susceptible S. aureus isolates resistant to erythromycin were resistant to clindamycin, indicating good correlation. Further, there were small numbers of methicillin-susceptible S. aureus isolates resistant to gentamicin, chloramphenicol, and quinupristin/dalfopristin.

In terms of multidrug resistance, as described above, all MRSA isolates were multidrug resistant, with USA100 isolates resistant to six to seven antimicrobial agents belonging to three subclasses and USA300 isolates resistant to seven antimicrobials in five subclasses. Table 5 lists the antimicrobial resistance profiles among the130 methicillin-susceptible S. aureus isolates from pork and beef. Among beef isolates, all except one were susceptible to all the antimicrobials tested, with the single isolate resistant to penicillin only. For pork isolates, 87 (74%) were resistant to at least two antimicrobial subclasses and 17 (14%) to at least four subclasses. The most commonly observed resistance profile was ampicillin–penicillin–tetracycline, occurring in 61 isolates (52%) from 28 pork samples (Table 5). There were small proportions of pork samples containing S. aureus isolates resistant to only one class of antimicrobial (5/47; 11%) or susceptible to all the antimicrobials tested (3/47; 6%; data not shown). Similar to SE profile, the majority (60%; 29/48) of meat samples containing multiple S. aureus isolates had the same resistance profiles for all isolates within the same sample (data not shown).

Table 5.

Antimicrobial Resistance Profiles of 130 Methicillin-Susceptible Staphylococcus aureus

	No. of antimicrobial subclasses resistant to	No. (%) of isolates with a specific profile		No. (%) of meats with a specific profile ^b
Antimicrobial resistance profile ^a	No. of antimicrobial subclasses resistant to	Beef (n = 12)	Pork (n = 118)	Beef (n = 5)	Pork (n = 47)
CLI-ERY-AMP-PEN-CHL-TET	5		3 (2.5)		1 (2.1)
CLI-ERY-AMP-PEN-SYN-TET	5		1 (0.8)		1 (2.1)
GEN-CLI-ERY-AMP-PEN-TET	5		2 (1.7)		2 (4.3)
CLI-ERY-AMP-PEN-SYN	4		3 (2.5)		1 (2.1)
CLI-ERY-AMP-PEN-TET	4		7 (5.9)		5 (11)
CLI-ERY-PEN-TET	4		1 (0.8)		1 (2.1)
CLI-ERY-TET	3		7 (5.9)		3 (6.4)
AMP-PEN-TET	2		61 (52)		28 (60)
PEN-TET	2		2 (1.7)		2 (4.3)
AMP-PEN	1		5 (4.2)		5 (11)
PEN	1	1 (8.3)		1 (20)
TET	1		15 (13)		10 (21)
Susceptible to all tested	0	11 (92)	11 (9.3)	5 (100)	6 (13)

Antimicrobial abbreviations are as following: AMP, ampicillin; CHL, chloramphenicol; CLI, clindamycin; ERY, erythromycin; GEN, gentamicin; PEN, penicillin; SYN, quinupristin/dalfopristin; TET, tetracycline.

Certain meat samples (1 beef and 15 pork) contained methicillin-susceptible S. aureus isolates with two resistance profiles and two pork samples contained S. aureus isolates with three resistance profiles.

Discussion

Studies conducted in many countries have indicated the presence of enterotoxigenic S. aureus in various food products and from food handlers, which are regarded as a primary contamination source leading to SFP (Seo and Bohach, 2007). For food products, the reported prevalence of enterotoxigenic S. aureus was 59.8% in meat and dairy products in Italy (Normanno et al., 2007), 62% in ready-to-eat foods in Korea (Oh et al., 2007), 74% in various food products in Poland (Lawrynowicz-Paciorek et al., 2007), and 69% in Portugal (Pereira et al., 2009). In contrast, much lower prevalence of enterotoxigenic S. aureus was also reported, such as 30.5% from various foods (cooked meals, meat, pasta, and cheese) in France (Rosec et al., 1997) and 39.2% from food samples (cheese, pasta, and sausage) and food manufacturers in Slovak Republic (Holeckova et al., 2002). For food handlers, surveys in multiple countries have revealed varied occurrence of enterotoxigenic S. aureus ranging from 21.1% in Botswana to 86.6% in Kuwait (Al-Bustan et al., 1996; Soares et al., 1997; Figueroa et al., 2002; Loeto et al., 2007; Udo et al., 2009). In comparison, the 85% overall prevalence of enterotoxigenic S. aureus isolates found in the present study was high. Additionally, 66% of S. aureus isolates harbored two to four SE genes, which is higher than 25.6% reported in an Italy study (Normanno et al., 2007) and 44.6% in Korea (Oh et al., 2007), but comparable to 61% in various foods in Portugal (Pereira et al., 2009). The difference in the types of foods examined and geographical locations may account for the differences in prevalence rates observed. In addition, many previous studies only examined classical SEs (Rosec et al., 1997; Holeckova et al., 2002; Normanno et al., 2007) and therefore may have underestimated the overall prevalence rate.

In the present study, the two major SE genes identified were seg and sei, each occurring in 66% of total S. aureus isolates examined, followed by seh (20%), sed (15%), and sej (13%). Only one isolate (1%) was positive for sea. The coexistence of seg and sei (Table 3) was not surprising because together with sem, sen, and seo, they belong to an enterotoxin gene cluster (egc) and the detection of one usually indicates the presence of others (Jarraud et al., 2001). The finding that seg and sei were predominant agreed with several previous studies examining S. aureus in foods or from food handlers (Lawrynowicz-Paciorek et al., 2007; Udo et al., 2009) but contrasted with others (Oh et al., 2007; Pereira et al., 2009), where sea was predominant. Analyzing S. aureus isolates from SFP cases indicated that SEA and SED were the two predominant SEs, followed by SEB (Balaban and Rasooly, 2000; Omoe et al., 2002; Cha et al., 2006; Kerouanton et al., 2007). However, such trend appears to change with time (Seo and Bohach, 2007). Although the involvement of newer SEs in SFP is not yet fully understood, it remains important to continue monitoring the changing epidemiology of SE or SE genes in foods and SFP cases.

Major SE gene profiles observed in this study in the descending order of predominance were seg-sei (50%), seh only (19%), and sed-seg-sei-sej (13%; Table 3). It is noteworthy that the sed-seg-sei-sej profile was only observed in 19 MRSA USA100 isolates. A very recent study characterizing MRSA isolates from patient with invasive disease found that for USA100, SED (74.5%) was the most frequently detected SEs among six SEs examined (SEA-SEE, SEH), whereas <1.5% isolates harbored SEA, SEB, or SEC (Limbago et al., 2009). Additionally, the same study reported that among 627 USA300 isolates, the only SE found was SED, occurring in <1% of isolates (Limbago et al., 2009). In the present study, the only SE gene found among USA300 isolates was seh in a single isolate, which agreed with the previous study regarding the low occurrence of SEs in USA300.

The detection of these SE genes in S. aureus isolates by PCR does not necessarily indicate the strains' capability to produce SEs in foods or the amount of SEs produced is sufficient to cause SFP. Omoe et al. (2002) reported that most seh-harboring S. aureus isolates would be capable of producing a significant amount of SEH; however, most of the S. aureus isolates harboring seg and 60% of isolates harboring sei did not produce detectable levels of SEG or SEI, although reverse transcription PCR proved that mRNAs of SEG and SEI were transcribed. Nonetheless, since immunoassays are only available to a limited number of SEs, PCR offers an alternative to detect S. aureus isolates with SE-producing potentials, especially in the case of multiplex PCR, which allows for the rapid and simultaneous detection of multiple SE genes.

Besides SEs, the only other exotoxin detected in this study was PVL in three MRSA USA300 isolates. In contrast, 9% of methicillin-susceptible S. aureus isolates from food handlers in Kuwait were PVL positive (Udo et al., 2009). Previously reported occurrence of TSST-1 in S. aureus isolates from foods or food handlers varied greatly (Oh et al., 2007; Gonano et al., 2009; Udo et al., 2009). In agreement with the present study, ETA and ETB were not detected in S. aureus from various foods in Korea (Oh et al., 2007).

Resistances to penicillin, ampicillin, tetracycline, and erythromycin were common in this collection of S. aureus isolates. The high resistance rates to penicillin and ampicillin concurred with several previous studies (Pereira et al., 2009; Udo et al., 2009). However, for tetracycline and erythromycin resistance, previously reported rates varied greatly (Soares et al., 1997; Loeto et al., 2007; Pereira et al., 2009; Udo et al., 2009). Among S. aureus food isolates in Portugal, resistances to erythromycin and tetracycline were 5% and 0.7%, respectively (Pereira et al., 2009), whereas among isolates from food handlers in Botswana, resistances to erythromycin and tetracycline were 18.6 and 50.5%, respectively (Loeto et al., 2007).

Multidrug resistance was common among MRSA isolates, which is not surprising due to their carriage of staphylococcal cassette chromosome mec elements and additional resistance determinants on plasmids (Zhang et al., 2005). Similar to previous findings, resistances to fluoroquinolones and macrolides were common among MRSA USA100 isolates, whereas resistance to macrolides was common among USA300 isolates (Limbago et al., 2009). For methicillin-susceptible S. aureus isolates, multidrug resistance was common among those isolated from pork (Table 5), whereas only one isolate from beef was resistant to single antimicrobial penicillin. This finding is interesting; however, due to the small number of beef isolates examined in this study, further large scale studies would be necessary to confirm this observation.

It is well known that the main risk arising from the presence of S. aureus in foods is the development of SFP. The high prevalence (85%) of S. aureus isolates with many (66%) harboring multiple enterotoxin genes found in this study highlighted the need for a better protection of foods from being contaminated during processing and handling. However, in the context of SFP, antimicrobial resistance is not relevant since SFP is not a disease that is treated with antimicrobials. The second risk of S. aureus in foods is developing invasive infections following the ingestion of contaminated food, but the likelihood is rather small (Kluytmans, 2010). Thirdly, there is a potential risk of becoming colonized with antimicrobial-resistant S. aureus, for example, MRSA, during food processing and handling (Kluytmans, 2010). The finding of antimicrobial-resistant S. aureus isolates harboring multiple SE genes, particularly MRSA with four SE genes, in the present study is worrisome since these isolates would pose a great risk of both food poisoning and colonization with antimicrobial-resistant enterotoxigenic S. aureus. Therefore, vigilant food safety practice needs to be implemented for people who handle raw meat products to prevent potential foodborne infections and intoxications due to S. aureus contamination.

Footnotes

Acknowledgment

We thank Dr. Jianghong Meng, University of Maryland, for suggestions on antimicrobial susceptibility testing method for S. aureus.

Disclosure Statement

No competing financial interests exist.

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