Development of Multiplex PCR for Staphylococcal Enterotoxin Gene typing and Detection of Enterotoxigenic Staphylococcus aureus

 

C. Anish, S. Ramesh^*, M. Muthusamy

Department of Microbiology, PRIST University, Thanjavur-613 403, Tamil Nadu, India

 

ABSTRACT:

Staphylococcus enterotoxins (SEs) are the main etiological agents of staphylococcal food poisoning. They were classified by serological criteria into 5 major groups and recently many more groups were identified. Staphylococcus enterotoxins (SEs) are pyrogenic toxins of the super-antigen family. Therefore to ensure food safety, it is important to detect and quantify the toxin genes to confirm the presence of enterotoxigenic S. aureus in any food samples. In this study, we have developed a multiplex PCR method to profile major toxin genes such as seB, seD, seE, seI and seM of Staphylococcus aureus using reference strains as well as some food isolates. The sensitivity of the single target and mixture of toxin gene targets were tested from five-fold serially diluted respective genomic DNA samples. The single PCR and multiplex PCR assays can successfully amplify the genomic DNA when used template as low as 4.5 pg/µl and 900 pg/µl respectively. Further these primers were validated on other species including target-negative reference strains as well as other gram positive/ negative reference strains. Among tested food isolates, 66% of them found to be positive for seB and 53% of the food isolates found to have seB and seE genes together. This method is highly suitable for convenient toxin gene profiling and to detect the presence of enterotoxigenic S. aureus.

 

 

 

INTRODUCTION:

Enterotoxin producing Staphylococcus aureus is one of the long known food-borne pathogens responsible for food-poisoning. The foods with improper processing normally found with enterotoxin produced by contaminating S. aureus species. The coagulase positive S. aureus normally can grow and produce enterotoxin (SE), a main etiologic agent of food poisoning quickly at room temperature in a wide variety of food materials such as dairy products, mixed foods, meat products, egg products, etc^[1]. S. aureus is a dessication tolerant organism with the ability to survive in potentially dry and stressful environments, which favors the growth of it in many food products^[2].

 

 

 

The concentration of 106 -108 CFU of S. aureus per gram of food or less than 1 g of enterotoxin is enough to produce food poisoning. S. aureus is one of the major issues threatening food industry since microbial contamination reduces shelf life and food quality apart from leading to food poisoning outbreaks and its consequences. Moreover SEs in foods is difficult to distinguish due to its lack of taste and food appearance.

 

Staphylococcus Enterotoxins (SEs) are pyrogenic toxins of the superantigen family due to their structural relatedness and biological activities. Based on the serological criteria SEs were classified by into five major groups A to E, referred to as SEA to SEE, respectively^[3]. Other studies reported the existence of other types, such as SEG, SEI, SEH, SEK, SER and etc. The recent researches spotted out that even some of the coagulase negative S. aureus are able to produce staphylococcal enterotoxins esp., sEA serological types^[4]. The microbiology based traditional detection methods are not reliable for specificity due to antigenically similar serotypes, moreover it is also a time consuming (5-6 days) procedures. It also depends upon other variables such as detectable levels of toxins etc^[4]. In the last 10 years, several PCR detection methods have been proposed for the detection of food-borne pathogens to replace the time-consuming culture-based classical techniques^[5-7]. They are very specific, rapid as well as sensitive and constitute very valuable tools for microbiological applications. Hence, molecular diagnostics methods such as PCR and real-time- PCR emerged as an effective alternative to traditional cultural methods and are highly recommended for detection of S. aureus.

 

Pathogenic S. aureus detection using PCR based methods were initially targeted many genes responsible for exfoliative toxins (ETs), and toxic shock syndrome toxin 1(TSST-1), β-hemolysin and also exotoxin producing genes such as SEA, SEB, SEC etc^[3]. However the recent reports claimed that more than 20 serotypes of S. aureus enterotoxins were identified to be found in food borne diseases which usually occurred with symptoms of nausea, vomiting, gastritis, diarrhea, loss of appetite, severe abdominal cramps, and mild to fatal fevers. Enterotoxin genes such as seA, B, C and D are predominantly found in most of the S. aureus species. Some of these enterotoxins are heat stable at higher temperatures hence it is not entirely possible to destroy entertoxins accumulated in food materials due to S. aureus contamination^[8]. Unlike other food borne illnesses, staphylococcal food poisoning occurs shortly after, 30 min to 8 hrs, food ingestion contaminated with enterotoxin. Intoxication is indicated via symptoms such as vomiting and diarrhoea resulting from the ingestion of foods accumulated with one or more enterotoxins.

 

PCR based detection methods especially focusing enterotoxin genes need to include specific primers for detection of these variants to avoid the false negative detection by sensitive molecular diagnostic methods like PCR or Real time PCR. Importance of specific probes targeting this antigenic variation has been understood earlier and research findings were being published^[6]. One or more variants of enterotoxin may present or not in single species of enterotoxigenic S. aureus. Hence it is relatively time consuming and tedious to employ individual PCR with specific probes to accurate detection. Alternatively, multiplex PCR (mPCR) can be employed especially where multiple fragments/regions of antigenic variants need to be amplified to confirm the presence of one microbial species in foods. In mPCR, multiple probes were designed to work at given reaction conditions to detect different loci or DNA fragments in single PCR. Although mPCRs for detection of few or several pathogens is rapidly rising and accumulated, multiplex for accurate detection of single pathogens especially enterotoxin produced by different strains of S. aureus is limited.

 

In the present investigation, we developed multiplex PCR for most predominant staphylococcal enterotoxins or serotypes such as SEB, SED, SEE, SEI and SEM. This study can be very useful to detect most of the enterotoxigenic species of S. aureus present in food or other samples.

 

MATERIALS AND METHODS:

Reference strains and isolation of other S. aureus strains from food materials:

A total of 45 strains of Staphylococcus spp. including 22 reference strains, 7 belonging to the species S. aureus and 15 to other species/genus, and 23 food isolates, were included in this study. Reference strains are equivalent to American Type Culture Collection strains (ATCC) were supplied by Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, INDIA. They were grown in Mannitol salt Agar (MSA) and UTI Agar (HiMedia, INDIA) at 37^oC for 48-72 hrs. Food isolates were recovered on Mannitol salt Agar incubated at 30^oC for 48-72 hrs and further grown on nutrient broth for genomic DNA extraction. 2 grams of each samples was suspended in 10 ml of sterile deionised water in case of solid food materials and 0.1 mL of 10^-1 dilutions was spread on the surface of Mannitol salt Agar. The plates were incubated for 48-72 hrs at 30ºC and typical colonies of coagulase-positive yellow colonies with yellow zone due to mannitol fermentation and non-fermenting coagulase-negative pink colonies were observed.

 

DNA extraction and multiplex PCR assay:

A 2-ml aliquot of overnight grown Staphylococcus aureus reference strains was collected for genomic DNA extraction. The genomic DNA extracted using cetyltrimethylammonium bromide (CTAB) a principle method with some modification and also QIAamp mini DNA kit (QIAGEN India, New Delhi) according to manufacturers instruction was used in this study^[9]. The concentration of isolated genomic DNA was measured using UV-spectrophotometer and was subsequently aliquoted in 0.5 ml microfuge tube and stored at-20⁰C until used. A total of five set of primers specific to five serotypes of Staphylococcal enterotoxin genes were employed to develop multiplex PCR in this study.

 

The mPCR assay was optimized using a Mastercycler nexus gradient thermal cycler (Eppendorf, USA) and the specificity of the each SE specific primers were confirmed using single target PCR. The negative control PCR reaction without DNA template was used to test for possible self-priming and nucleic acid contamination. A total 20 µl of mPCR mixture consisted of 12 µl of Emerald green dye Master mix (TaKaRa Clontech), five primers each with 10 pM concentrations, 45 ng of each target positive template DNA mixture (ng for each primer) and nulcease free water to final volume. The mPCR was optimized to temperature profile of 94⁰C for 5 min followed by 35 cycles of 94⁰C for 30 s, 61⁰C for 30 s and 72⁰C for 30 s with a final extension cycle at 72⁰C for 5 min. The PCR amplicons were resolved in a ethidium bromide (10 mg/ml) added 3% of agarose gel at 100 V for 45 min and the gel images were captured using GELSTAN documentation system.

 

Specific detection of S. aureus strains from spiked milk and bread samples:

Sensitivity assays were carried out on spiked milk and bread samples prepared as follows: 0.01 ml of overnight grown cultures of S. aureus were inoculated in 10 ml of pasteurized milk. In case of solid food material (bread slices 2gm ca) was initially homogenized with 10 ml sterile water/ broth and then inoculated with a loopful of inoculum (0.01ml) and kept for 24-48 hrs. The negative controls were maintained without inoculation. After enrichment, 2 ml of cultures were taken for DNA extraction using Qiagen DNA extraction kit.

 

Sensitivity Assay for single and multiplex PCR:

To determine the sensitivities of the single and multiplex PCR assays, the minimal amount of staphylococcal DNA that would be successfully detected was determined. Purified DNA of reference strains was quantified using a spectrophotometer. Initially, 450ng/µl was tested with PCR, and then 5-fold serial dilutions (45ng, 4.5ng, 0.45ng or 450pg, 45pg and 4.5pg) of ending at 4.5pg/µl were subsequently used as template for single PCR and 9.0pg/ µl was used for multiplex PCR. Upon completion of PCR, an aliquot (10µl) from each tube was analyzed by electrophoresis. To compare the influencing factors of multiplex procedure, where mixture of 5 set of primers used versus that of any one primer with single-PCR, DNA of the toxin-positive reference strains was tested as a single target versus equal amounts of a mixture of all 5gene targets.

 

To determine the specificity of PCR, samples of DNA from the target toxin-negative S. aureus strain (737) and from other gram positive/negative reference strains were tested. 45 ng of template DNA was used per reaction.

 

RESULT AND DISCUSSION:

All reference strains as well as food isolates were used in this study and their genomic DNA was isolated either using CTAB^[9] or QIAamp mini DNA kit. The isolated genomic DNAs were resolved by 0.8% agarose gel electrophoresis and then their concentration for PCR was determined using spectrophotometer. The PCR conditions for all designed primers seB, seC, seD, seE, seI and seM (Table 1) were standardized with reference strains prior to screening of enterotoxin genes of food isolates. To favour the multiplex PCR, melting temperature (Tm) of all primers were more or less similarly designed and synthesized around to be 58-60⁰C. Among enterotoxin genes seB, seC, seD, seE, seI and seM were tried and multiplex PCR was successfully adopted for all genes except seC. All five primers in single and multiplex PCR reactions successfully amplified the appropriate regions of the target toxin genes from a DNA dilution containing as little as 4.5 pg for single PCRs and in multiplex PCRs as little as 900 pg of DNA of a DNA mixture isolated from reference strains containing the test toxin genes (Fig.3). The DNA of one or more exotoxin-producing reference strains of S. aureus was specifically amplified by the primers. The sizes of the amplicons were same as the expected product of the designed primers (Fig. 1).

 

 

Fig 1 shows the representative gels for the PCR product patterns of 7 different strains of reference S. aureus. M depicts the band pattern of 50 bp DNA ladder and lane1-7 presents PCR product of reference strains of S. aureus MTCC-737 (ATCC6538P), MTCC-3750, MTCC-1430 (ATCC12600), MTCC-96 (ATCC9144), MTCC-902 (ATCC12598), MTCC-435 (ATCC155), and MTCC-3103 respectively.

 

In addition, none of the primer pairs reacted with any DNA of other species or genus such as few strain of Escherichia coli, Bacillus cereus (MTCC-430/ATCC11778), Shigella flexneri (MTCC-1457/ATCC29508), Shigella boydii (MTCC-11947/ATCC 8700) Salmonella typhimurium (MTCC-3224), Salmonella enterica (MTCC-3219) and Yersinia enterolitica (MTCC-861/ATCC9610), Yersinia intermedia (MTCC-3101) were tested in this study. However, coagulase negative strain Staphyloccocus epidermis was positive for all enterotoxin gene except seE. From the study we identified that almost all 6 reference S. aureus were positive for enterotoxin genes although band intensities varied between them. Hence the slight band found expected size of amplicons not found in negative controls without templates, they were considered as Positive for enterotoxins. Visibly prominent bands for amplicons found for seB in MTCC-96 (ATCC9144), MTCC-902 (ATCC12598) and seC in all 6 strains except MTCC-96 (ATCC9144), seD in MTCC-1430 (ATCC12600), MTCC-96 (ATCC9144), seE in MTCC-737 (ATCC6538P), MTCC-1430 (ATCC12600), MTCC-96 (ATCC9144), MTCC-902 (ATCC12598), and MTCC-3103, seI and seM in MTCC-737 (ATCC) and MTCC-3103 respectively.

 

 

Fig 2 PCR product pattern of 5 set of primers for enterotoxin genes such as seB, seD, seE, seI and seM in multiplex PCR. The amplicons were resolved by 3% High resolution Agarose gel electrophoresis in TAE buffer.

 

In a given multiplex PCR mixture consisting of DNA from every type of target gene, the resulting band intensities of the larger amplicons esp. seB, seD and seM were reduced while intensities of amplicons such as seE, seI were almost conserved in multiplex PCR similar to single PCR reactions.

Table 1 Primers used for the detection of enterotoxigenic Staphylococcus aureus strains

Primer Name	Sequence (5’-3’)	Product (bp)	Target gene
entB-F	GTT CGC CTT ATG AGA CTG GCT A	88	seB
entB-R	TTT TCA CCA GAT TCA GGC ATC AT
entC-F	TGG GAA TGT TGG ATG AAG GAG A	127	seC
entC-R	TAG GGT CTG GTT GGC TCT CT
entD-F	TGT GTA AGA AGT CAA GTG TAG ACC	312	seD
entD-R	TTA CCC CAC CAT ATG TAC AGG C
entE-F	GTT GTA CGA ACA AGG TGG CGA	113	seE
entE-R	CCT CAT GAC CAT ACT CAC CAT TT
entI-F	GGT GGA GTC ACA GCT ACT AAC G	145	seI
entI-R	TGA CAT CAA TCT CTT GAG CGG
entM-F	GCG CTC AAG GCG ATA TAG G	263	seM
entM-R	GTT AGT AGC TGT GAC TCC ACC AT

 

Sensitivity Assay:

 

Fig 3 Evaluation of single and multiplex PCR detection limits using all 5 toxin positive Staphylococcus aureus genomic DNA mixture. Genomic DNA concentration ranging from 450 ng to 4.5 pg and 900ng to 9.0pg was used as template in single target and multiplex PCR assay. The single and multiple amplicons were resolved by 3% AGE.

 

All five primers in single and multiplex PCR reactions successfully amplified the appropriate regions of the target toxin genes from a DNA dilution containing as little as 4.5 pg for single PCRs and in multiplex PCRs as little as 900 pg of DNA of a DNA mixture isolated from reference strains containing the test toxin genes (Fig 4). When 90 pg of mixture of target positive genomic DNA used, multiplex PCR failed to produce anyone or more amplicons.

Table 2. List of Staphylococcus aureus food isolates and their profile of different enterotoxin gene from contaminated ready to use Indian food items.

S.No	Isolates	seB gene	seE gene	seD gene	seI gene	seM gene
1.	ATCC6538P	+	+	+	+	+
2.	MTCC-3750	+	-	+	+	+
3.	ATCC12600	+	+	+	+	+
4.	ATCC9144	+	+	+	+	+
5.	ATCC12598	+	+	+	+	+
6.	ATCC155	+	-	+	+	+
7.	MTCC-3103	+	+	+	+	+
8.	Coco-FS-1	+	+	-	+	-
9.	WNF-1	-	-	-	-	-
10.	SAM-NF-2	-	-	-	-	-
11.	SAM-NF-3	-	-	-	-	-
12.	Coco-FS-2	-	+	-	-	-
13.	WNF-2	-	+	-	-	-
14.	L-SF-1	-	+	-	-	-
15.	M-L-F	+	+	-	-	-
16.	M-Pink	+	-	-	-	-
17.	C-UTI-Pink	-	-	-	-	-
18.	T-UTI-Pink	-	-	-	-	-
19.	Chic-NF	-	-	-	-	-
20.	FruitM-F-1	+	+	-	-	-
21.	1Milk-F	+	+	-	-	-
22.	1Milk-NF	+	+	-	+	-
23.	C-Chic-F	+	+	-	-	-
24.	C-Chic-NF	-	-	-	-	-
25.	Milk-F	+	+	-	-	-
26.	MeatS-Sl-F	+	+	-	-	-
27.	2FSA-1	+	+	-	-	-
28.	2FSA-2	+	-	-	-	-
29.	2SF-1	+	+	-	-	-
30.	EGG-1-F	+	+	-	-	-

 

 

 

We have evaluated 30 isolates including 7 reference strains for enterotoxin gene using mPCR while almost all the reference strains were harboring 5 enterotoxin genes such as seB,E,D,I and M. However the food isolates were mostly positive for only seB and seE genes. Among 30 strains tested, 66 % of the strains were possessing seB and 63% of the isolates were positive for seD, and followed by seI (30%) gene. Interestingly 53% of the food isolates found to have seB and seE enterotoxin genes together in this study. Previously seB, seD and seE were reported in food-borne disease outbreaks in various countries^[2]. Although multiplex PCR was initially designed for detection of SEE genes in S. aureus, from our study it is essential to design detection strategy by including seB along with other enterotoxin genes since seB found to be predominant in our study. Next to seB and seE, seI genes found predominant than seD and seM. Therefore we corroborates the findings of Argudin et al^[10] that new enterotoxin gene must be included to classical enterotoxin genes in molecular diagnostics of enterotoxigenic Staphylococcus aureus isolates in food and clinical samples. Omoe et al^[11] also reported that new types such as SEI and SEM also contributed to food poisoning outbreaks and further he suggested the importance of detection methods to include the new types of SE genes in detection of enterotoxigenic S. aureus detection in food borne diseases. Earlier, mPCR analysis identified 9, 11 and 4% of cheese and meat products for positive to the presence of enterotoxin such as seA, seD and SEE respectively^[12-16]. In our study seB and seE found in tested 2 meat samples although the number of samples should be more to conclude the distribution of types of enterotoxin genes. In another food borne disease outbreak in Taiwan reported that 11% of the samples tested were contributed by the presence of S. aureus harboring SEM genes^[12]. However, no incidence of seM amplicons found in 23 ready to use contaminated food isolates. Based on the previous reports^[4] that even coagulase-negative staphylococcal strains produce enterotoxin genes, we included some coagulase-negative food isolates based on the colony morphology i.e. pink colonies due non-fermentation of mannitol present in the MSA medium were also tested for enterotoxin genes^[17-22]. Of 8 isolates tested, 7 of them found to have no enterotoxin genes. Each one isolates from milk and mango harboring seB, E, I and seB genes respectively indicating rare coagulase-negative staphylococcal strains too might cause similar symptoms as like that coagulase-positive strains.

 

We have evaluated enterotoxigenic S. aureus genotypically from ready use or prepared food items because the screening for contamination of these species is usually lacking. Interestingly it was believed that some of the traditional food items having lot of spices expected to inhibit growth of contaminating bacterial growth. But our study showed that enterotoxigenic S.aureus can grow even red chilly mixed meat products if contaminated. It was suggested that the variation of S. aureus isolates and its ability to produce enterotoxin depends on the source/ food materials. Liquid foods showed high presence of enterotoxigenic species of S. aureus. We have shown that the multiplex PCR assay cab be a reliable method of accurately identifying the different enterotoxin gene producing S. aureus species and also can be used to distinguish enterotoxigenic and or coagulase-positive S. aureus from coagulase-negative S. aureus since very rarely coagulase-negative species produce enterotoxin^[12-15]. Most of the mannitol fermenting presumptive coagulase-positive S. aureus colonies (Yellow/white colonies with yellow zone) was harboring at least one enterotoxin gene reported here. It can be used as an alternative method for the routine microbiological analysis and the treatment can be fastened as this assay has only a short turnaround time. Though the naturally occurring food samples tested in the present study are low in number, the results obtained are highly promising and need to be evaluated further on a larger and diverse food sample sources.

 

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Accepted on 03.08.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(10): 4343-4348.