Phenotypic Characterization and Prevalence of Enterotoxin Genes in Staphylococcus aureus Isolates from Outbreaks of Illness in Chengdu City

Abstract

Staphylococcus aureus produces a spectrum of enterotoxin that is recognized as the main reason for causing staphylococcal food poisoning. The aim of the current study was to investigate the phenotypic characteristics and enterotoxin genotypes of S. aureus isolated from food poisoning sufferers. On the basis of the amplification of 16S rRNA and nuc gene specific to S. aureus assay and the phenotype (hemolytic activity, thermal stable nuclease [Tnase] test, and biofilm formation), all isolates were identified as S. aureus. To genotypically characterize S. aureus isolates, genes encoding staphylococcal enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sem, sen, ser, and seu) were investigated by using polymerase chain reaction technique. The results showed that the eight isolates of S. aureus had different enterotoxin genotypic characteristics, which was the main cause of food poisoning. One isolate contained 10 enterotoxin genes, and the other 7 isolates carried 3 or more enterotoxin genes. The frequency of the newly identified enterotoxin genes (seg-seu) was higher than classical genes (sea-see). Overall, multi-gene detection rates were 75% (for sek, ser, and seu); 50% (for sea and sem); 37.5% (for sen, seg, and sei); and 12.5% (for seb, sec, sed, and sej), respectively. The see and seh gene were not detected in any isolates. The current study provided the exact distribution of enterotoxin genes in eight S. aureus strains from food poisoning sufferers, which indicated that the pathogenicity of the newly identified enterotoxin should be highlighted. The need for prevention of food poisoning occurrences caused by enterotoxin of S. aureus should be reinforced.

Introduction

S taphylococcal food poisoning is one of the most common foodborne diseases worldwide resulting from the ingestion of staphylococcal enterotoxin (SE) preformed in food with high protein content by enterotoxigenic strains of Staphylococcus aureus (Ostyn et al., 2010). To date, SEs have been divided into five serological types (SEA through SEE) on the basis of their antigenicities and other new types (SEG, SEH, SEI, SEJ, SEK, SEM, SEN, SER, and SEU), which share structure and sequence similarities (Thomas et al., 2007; Ono et al., 2008; Casagrande Proietti et al., 2010). The SE is heat-stable and low-molecular protein (MW 26,900–29,600), which may be present in food when S. aureus strains are absent (Omoe et al., 2002). After ingestion of contaminated food, toxins are resorbed into the blood in the gastrointestinal tract, activating an emetic reflex, causing nausea, emesis, abdominal cramps, and diarrhea (Holecková et al., 2002). Therefore, a conclusive staphylococcal food poisoning diagnosis is mainly based on the detection of SE.

It is estimated that about 95% of staphylococcal food poisoning outbreaks are caused by SE types SEA to SEE. The remaining 5% of outbreaks may be associated with other newly identified SE (Omoe et al., 2002). In fact, there is little information concerning the occurrence and significance of the novel enterotoxin in food poisoning outbreaks. The relationship between these new SE and human food poisoning is not fully understood at present. Moreover, great efforts have been applied to the development of detection methods for classical SE. The newly identified enterotoxin cannot be detected by the commercial detection system. Obviously, the detection of classical and newly identified enterotoxin genes is equally vital for the analysis of staphylococcal food poisoning. In the current study, to investigate the classical and newly identified SE genes, we used polymerase chain reaction (PCR) to detect and analyze the distributions of the sea to seu genes in eight S. aureus isolates obtained from humans involved in food poisoning outbreaks in Sichuan, China.

Materials and Methods

Bacterial strains

S. aureus isolates (1–8); S. aureus ATCC25923; S. aureus ATCC6538; each enterotoxin positive control of S. aureus (containing sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sem, sen, ser, and seu genes, respectively) was isolated from different sources, established, and preserved in our laboratory.

S. aureus strains identification from food-poisoning cases

S. aureus isolates from one of the community hospital of Chengdu city, Sichuan, China, were isolated from the vomit and fecal specimens of individual patients associated with staphylococcal food-poisoning outbreaks that occurred from April to July 2010. A total of eight cases were consecutively enrolled. The epidemiological data concerning these outbreaks (number of cases, symptoms, location, and types of potentially incriminated food) were collected by the community hospital. Meat-based dishes were found to be the common source of the outbreaks. These strains were identified according to the methods described in GB 4789.10-2010 Food microbiological examination: S. aureus. In general, test strains were cultured in tryptic soy broth, Baird-Parker (BP) agar, and tryptic soy agar followed by Gram staining, microscopic examination, hemolytic activity (determined on blood agar), thermal stable nuclease (TNase) test (using Toluidine blue agar), and biofilm formation assay (on glass surface). Further, all isolated strains were investigated and identified to the species level by PCR. The presence of the target 16S rDNA (Staphylococcus genus specific) and nuc gene (S. aureus species specific) was determined to be S. aureus species (Tang et al., 2006).

Extraction of genomic DNA

Genomic DNA used for PCR analysis was isolated from the eight strains by the extraction method described by Tang et al. (2006).

Specific primers

The staphylococcal classical enterotoxin genes primer sets (A1/A2, B1/B2, C1/C2, D1/D2, and E1/E2) were derived from Wang et al. (2002). The new enterotoxin genes primer pairs were designed according to the S. aureus enterotoxin G, H, I, J, K, M, N, R, and U gene sequences (GI:58397435; GI:510691; GI:110681906; GI:3372540; GI:255506223; GI:58397427; GI:146747401; GI:37196677; and GI:37681489, respectively) by using the Primer Premier 5.0 software (PREMIER Biosoft). The primer pairs were also aligned by using Basic local alignment search tool (www.ncbi.nlm.nih.gov/BLAST). The PCR primers and conditions for seg, seh, sei, sej, sek, sem, sen, ser, and seu genes amplification were listed in Table 1. Molecular sizes for the sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sem, sen, ser, and seu amplified products were 582, 732, 403, 251, 474, 592, 431, 345, 476, 271, 318, 301, 526, and 243 bp, respectively.

Table 1.

Primers for Detection of Staphylococcal Enterotoxin Genes

Enterotoxin	Primers	Primer sequences (5′-3′)	Amplicon size (bp)	References
SEA	A1	ATTAACCGAAGGTTCTGTAGA	582	Wang et al. (2002)
	A2	TTGCGTAAAAAGTCTGAATT
SEB	B1	TTTTTCTTTGTCGTAAGATAA	732	Wang et al. (2002)
	B2	CCAACGTTTTAGCAGAGAAG
SEC	C1	TAAGTTCCCATTATCAAAGTG	403	Wang et al. (2002)
	C2	CCAACGTTTTAGCAGAGAAG
SED	D1	TAATGCTATATCTTATAGGG	251	Wang et al. (2002)
	D2	TTGCGTAAAAAGTCTGAATT
SEE	E1	TAAACCAAATTTTCCGTG	474	Wang et al. (2002)
	E2	TTGCGTAAAAAGTCTGAATT
SEG	G1	CTGAATAAGTTAGAGGAGGTTT	592	This study
	G2	TCTTTAGTGAGCCAGTGTCTTG
SEH	H1	ACTATCATTTCCATTCTAACTAC	431	This study
	H2	AATCATTGCCACTATCACCTTG
SEI	I1	TATGTATGGAGGGGTCACTTTATCA	345	This study
	I2	AAGGAACTTACAGGCAGTCCATCTC
SEJ	J1	TAACCCCTTTAGTTTACAGCGATAG	476	This study
	J2	AATGGTTACTTTTTTCTTATTTGTT
SEK	K1	ATGGATCAATGGAAATCACAAA	271	This study
	K2	TGGTAACCCATCATCTCCTGTG
SEM	M1	TTACTTTAGCGGGTGATT	318	This study
	M2	CTTGCCCTGTTCCTGTAT
SEN	N1	TAAACGGAGGAGTTACGATA	301	This study
	N2	AACTCTGCTCCTACTGAACC
SER	R1	ATAAAGCGGTAATAGCAGAA	526	This study
	R2	GGTATAAAGGGAACCAAATC
SEU	U1	AAACATTAAAGCCCAAGAG	243	This study
	U2	ACACCGCCATACATACAC

SE, staphylococcal enterotoxin.

Detection of SE genes

After the DNA extraction, amplifications of enterotoxin genes were performed by PCR. The following 14 enterotoxin genes (including sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sem, sen, ser, and seu) were examined. Reactions were carried out in a volume of 20 μL with a 10×PCR buffer, 2 μL; 2.0 mM MgCl₂; 200 μM of each nucleotides dATP, dTTP, dGTP, and dCTP; 1 U of Taq polymerase [from TaKaRa Biotechnology (Dalian) Co., Ltd.], 1 μL of purified sample DNA, and with primers 0.5 μM. The mixes were submitted to a program performed on a thermocycler (Mycycler; BioRad) with an initial denaturation step at 95°C for 5 min, 35 amplification cycles each with 40 s at 95°C; 50 s at 48°C (for seh) or 52°C (for sea, seb, sec, sed, see, sej, and seu) or 54°C (for sek, sem, and ser) or 55°C (for seg, sen) or 58°C (for sei); and 50 s at 72°C followed by an additional extension step of 10 min at 72°C.

Results

Phenotypic characterization and identification of S. aureus isolates

Preliminary estimation for the microbial isolates was carried out by cultural properties on BP agar. Staphylococcal nuclease activities of all the isolates were detected on toluidine blue-DNA agar. The presence of pink halo showed that all the isolated strains could produce the staphylococcal nuclease enzymes. Hemolytic activities were determined on blood agar, and the tested S. aureus strain No.5 presented strong hemolysis on blood agar plates. The capacity for biofilm formation could be observed on a glass surface with different S. aureus isolates. Strains No. 1, 4, and 8 could form biofilm. Further, species-specific PCR amplification showed that all the isolates were identified as belonging to S. aureus, which generated the detectable fragments of 223 bp (nuc gene) and 565 bp (16S rDNA gene) in 1% agarose gel.

Distributions of sea to seu genes in S. aureus isolates

The results of the PCR analysis were shown in Table 2. Overall, 14 SE genotypes were observed. Among all the outbreak-related isolates, 8 (100%) were found to be positive for SE genes; 5 (62.5%) possessed the sea, seb, sec, or sed gene; and 8 (100%) harbored the seg, sei, sej, sek, sem, sen, ser, or seu gene. Considering the classical SE genes, four (50%) isolates had the sea gene; one had seb; one had sed; and one had sec. The see gene was not found in any of the isolates. With regard to the new SE genes, three isolates had seg; three had sei; one had sej; six had sek; four had sem; three had sen; six had ser; and six had seu. The see gene and seh gene were not found in any of the isolates. All the isolated strains contained at least three types of SE genes; 10 SE genes were detected especially in strain No. 3. Taken together, the detection rate of the newly recognized SE genes (seg through seu) was higher (eight of eight PCR-positive strains) than that of the classical SE genes (sea through see, five of eight strains). Comparatively, the frequency of individual SE gene was variable and was as follows: sek=ser=seu>sea=sem>seg=sei=sen>seb=sec=sed=sej>see=seh.

Table 2.

The Profiles of 14 Staphylococcal Enterotoxin Genes in 8 Outbreak-Related Isolates of Staphylococcus aureus

	Isolated strain no.
SE genes	1	2	3	4	5	6	7	8	Total	Detection percentage
sea	+		+				+	+	4	50
seb	+								1	12.5
sec		+							1	12.5
sed			+						1	12.5
see									0	0
seg		+	+	+					3	37.5
seh									0	0
sei	+	+	+						3	37.5
sej			+						1	12.5
sek	+		+		+	+	+	+	6	75
sem			+	+	+			+	4	50
sen		+	+	+					3	37.5
ser	+		+	+		+	+	+	6	75
seu		+	+	+	+	+		+	6	75
Total	5	5	10	5	3	3	3	5

Discussion

Gastroenteritis is one of the most frequent microbial diseases that is caused by the ingestion of food contaminated with SE (Holecková et al., 2002). Classical enterotoxin SEA to SEE has been clearly demonstrated as being capable of more or less potent emetic activity. However, the majority of newly recognized SE has not been functionally characterized (Morandi et al., 2009); merely SEG, SEH, and SEI have been reported as having the ability to cause emetic response (Le Loir et al., 2003; Letertre et al., 2003; Bania et al., 2006). From this finding, the other newly recognized enterotoxin could also have the capability of causing the food poisoning symptoms, which are reflected in Table 2 with isolates 4, 5, and 6. These strains were detected to merely contain the newly recognized enterotoxin genes: sek, sem, sen, ser, and seu. To our knowledge, these new enterotoxins have not been reported as involving in food poisoning.

Recent studies have investigated the prevalence of SE genes in S. aureus strains from geographically diverse locations (Ruimy et al., 2009; Varshney et al., 2009). Food poisoning attributed to S. aureus accounts for about 25% of foodborne illnesses in China, whereas in some area, it accounts for 40% of foodborne illness. S. aureus types A, B, C, D, and E have been isolated with varying frequencies; typically, the majority of S. aureus isolates are types A and B, followed by types C and D. In this study, sek, ser, and seu (75%) were the most prevalent SE in all the isolates. Relatively higher rates of prevalence (50%) were noted for sea and sem genes. The frequency of the newly identified enterotoxin genes (seg-seu) was higher than that of the classical genes (sea-see). The results confirmed the fact that the frequency of potentially enterotoxigenic S. aureus would dramatically increase considering the newly detected seg-seu genes with the classic sea-see genes.

According to the results of the current study, we confirmed that all the eight strains were SE-producing strains, which were major factors causing the patients serious staphylococcal food poisoning. Our results provided the accurate distribution of the genotypes of enterotoxin in S. aureus isolates from patients associated with staphylococcal food poisoning, which should contribute to provide updated data on the carriage of SE genes in clinical isolates.

Footnotes

Acknowledgments

This work was jointly supported by the National Natural Science Foundation of China (No.31071515); the Special Foundation for Young Scientists of Sichuan Province, China (No. 2011JQ0043); and the Project for Standardization and Sharing of Vet Microbial Resources in Sichuan Province.

Disclosure Statement

No competing financial interests exist.

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