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. 2015 Autumn;16(4):381–384.

Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz

R Moghassem Hamidi 1, S Hosseinzadeh 2,*, S S Shekarforoush 2, M Poormontaseri 2, A Derakhshandeh 3
PMCID: PMC4782680  PMID: 27175208

Abstract

The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended.

Key Words: Coagulase, Enterotoxin, Staphylococcus aureus, Thermonuclease

Introduction

The major characteristics of staphylococcal enterotoxins (SEs) are low molecular weight, resistance to heat, pepsin digestion and superantigenicity (Bergdoll et al., 1979 ▶; Klotz et al., 2003 ▶) Symptoms of SEs include increased saliva, vomiting, abdominal cramping and diarrhea which can be accompanied by blood in some cases (Gómez et al., 2007 ▶). Approximately 5% of food poisoning illnesses are estimated to occur by staphylo-coccal enterotoxins (Vimercati et al., 2006 ▶).

Various types of Staphylococcus aureus enterotoxins including SEA, SEB, SEC1, SEC2, SEC3, SED and SEE are currently recognized (Morandi et al., 2007 ▶). Most strains produce one or more enterotoxins (Pourmand et al., 2010 ▶). Of the numerous physiochemical characteristics of S. aureus used for its classification, production of coagulase and thermonuclease is the most reliable practical criterion used in identifying the bacteria (Brakstad et al., 1992 ▶).

Enterotoxin A is the most common type among food-related strains of Staphylococcus (Barati et al., 2006 ▶). Several studies have shown that 15% to 80% of S. aureus isolates from various sources were able to produce enterotoxin (Omoe et al., 2005 ▶; Bania et al., 2006 ▶). The present study was aimed to investigate the correlation between enterotoxin A gene, producing enterotoxin A protein and coagulase and thermonuclease enzymes in S. aureus isolated from various sources in Shiraz.

Materials and Methods

Samples were collected from different sources including human pus, mastitic milks, and foodstuffs in order to identify S. aureus using the bacteriological standard method. Briefly, each sample was cultured on Baird Parker agar (Merck, Germany) and incubated at 35°C for 36 h. The suspected black colonies were further analyzed using Gram stain and biochemical tests (Quinn et al., 2002 ▶).

Coagulase and thermonuclease tests were performed, as was previously described. The presence of thermo-nuclease was confirmed based on the formation of a clear zone around the colonies (Baron and Finegold, 2013 ▶).

DNA extraction was performed using Cinnapure DNA kit (Cinnagen, Iran) based on manufacturer’s instruction.

PCR was performed with a final volume of 25 µL containing final concentrations of 50 mM KCl, 10 mM Tris-HCl (pH = 8.3), 1.5 mM MgCl2, 200 µM of each deoxynucleoside triphosphate (dATP, dTTP, dGTP, and dCTP), 25 pmol of each primer (Brakstad et al., 1992 ▶; Mehrotra et al., 2005 ▶; Ahmadi et al., 2010 ▶) and 1 U of Taq DNA polymerase (Vivantis, Malaysia). The amplified products were electrophoresed through a 1.5% agarose gel and visualized by staining using ethidium bromide.

Improving technique for gel electrophoresis of unknown protein using bacteriophage T4 was firstly established by Laemmli (1970) ▶ in which, four major protein components of the head of the bacteriophage was firstly recognized. The method was then extensively used worldwide.

Correlation between the presence of enterotoxin A gene and the production of coagulase and thermo-nuclease enzymes, was investigated using a Spearman’s rho correlation and Chi-square analysis. The statistical differences were considered significant when the p-values were equal and/or less than 0.05.

Results

One hundred S. aureus strains were isolated from human pus (n=40), mastitic milk (n=30) and foodstuffs (n=30) andconfirmed by biochemical test. All the strains were positive for the thermonuclease test. For the coagulase test, 54% of them presented a strong reaction (+3), while 46% were considered +1 or +2 (Table 1).

Table 1.

Frequency of Coa, Nuc and Sea gene in S. aureus

Source Number Coagulase +1 Coagulase +2 Coagulase +3 Coa gene Nuclease test Nuc gene Sea gene Expression of Sea gene
Human 40 6 11 23 36 (90%) 40 40 (100%) 9 (22.5%) 7/26
Animal 30 4 11 15 28 (93.3%) 30 30 (100%) 0 (0%) 0/13
Food 30 5 9 16 27 (90%) 30 30 (100%) 5 (16.6%) 4/16
Total 100 15 31 54 91 (91%) 100 100 (100%) 14 (14%) 11/54
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The frequency of Coa gene in the isolated strains of human, animals and foodstuffs was 90%, 93.3% and 90%, respectively (Fig. 1). The PCR assay confirmed the Nuc gene in all isolates (Fig. 2), whereas the Coa gene was detected in 91 strains which were phenotypically considered as 1+ (Table 1).

Fig. 1.

Fig. 1

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Representation of PCR product of Coa gene. Lane 1: Marker 1000 bp, Lane 2: Negative control (no template), and Lane 3: Coa gene 720 bp

Fig. 2.

Fig. 2

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Representation of PCR product of Nuc gene. Lane 1: Marker 1000 bp, Lane 2: Negative control (no template), and Lane 3: Nuc gene 397 bp

The prevalence of the enterotoxin A gene was 14% (human samples: 22.5%, food samples: 16.6%), but it was not found in any of the animal isolates. Moreover, the results showed that the occurrence of the enterotoxin A in animal samples was significantly lower than that in the human and food isolates (P<0.05) (Fig. 3, Table 1).

Fig. 3.

Fig. 3

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Representation of PCR product of Sea gene. Lane 1: Marker 1000 bp, Lanes 2, 4: Negative control (no template), and Lane 3: Sea gene 102 bp

The expression of Sea using SDS-PAGE analysis gene was confirmed in 11 strains (Fig. 4). As such, 78.6% of enterotoxin A genes were expressed.

Fig. 4.

Fig. 4

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SDS PAGE analysis of enterotoxin on a 10% polyacrylamide gel. Lane 1: Marker (KDa), Lane 2: Negative control, and Lane 3: SEA protein

Statistical correlation of the genes

No correlation was shown among the presence of enterotoxin A gene and Coa gene (P=0.21) and production of the coagulase (P=0.17) (Table 2).

Table 2.

Correlation between various tests and presence of Coa and Sea genes

Specific test Source Coa gene Sea gene
Coagulase test P=0.88, R=-0.015 P<0.001, R=0.453 P=0.09, R=0.169
Coa gene P=0.95, R=-0.006 – P=0.21, R=0.127
Sea gene P=0.32, R=-0.101 – –
Sea gene expression P=0.65, R=-0.064 – P<0.001, R=0.85
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Discussion

The present study revealed a prevalence of 22.5% enterotoxin A gene among the human isolates. Previous studies using different techniques showed the prevalence of 15%, 12% and 46.9% of Sea gene in Jordan, Germany and Iran, respectively (Klotz et al., 2003 ▶; Naffa et al., 2006 ▶; Pourmand et al., 2010 ▶).

Former studies have shown variability in the enterotoxin gene prevalence in S. aureus isolated from mastitic milk (Ahmadi et al., 2010 ▶). In Italy, Vimercati et al. (2006) ▶ reported more than 70% enterotoxin prevalence in S. aureus isolates recovered from cows with mastitis (Vimercati et al., 2006 ▶). 85 of the isolates (73%) harbored at least one enterotoxin gene (Se) with a predominance of Sea, Sed and Sej among isolates from bovine. In the current study, none of our isolates showed the occurrence of Sea gene. Our results were supported by the work performed by Gomez et al. (2007) ▶, in which none of Sea, Seb and Sec was identified from the S. aureus isolates.

In this study, prevalence of Sea among 30 food samples was 16.7%. Many studies have been carried out on the detection of enterotoxin gene in food. Asao et al. (2003) ▶ reported a food poisoning outbreak resulting from the consumption of milk and yogurt made from milk contaminated with enterotoxin A in Japan. The prevalence of 15.6% of Sea gene among S. aureus isolated from dairy products was reported in Tehran (Imanifooladi et al., 2010 ▶). According to the study of Ertas et al. (2010) ▶, the highest frequency of Sea gene among different foods was demonstrated. Attention to the hygiene condition during production and use of heat process is critical. As shown, PCR method was able to identify potential strains to produce enterotoxin (Najera-Sanchez et al., 2003 ▶). Gene expression assessment in isolated strains using SDS-PAGE showed 11 out of 14 strains contained enterotoxin A gene. The difference is possibly associated with the lack of expression of some genes. Furthermore, some strains produce lower level of enterotoxin below the sensitivity of phenotypic method. No significant correlation was observed between presence of enterotoxin A gene and production of coagulase and thermonuclease enzymes. In the study conducted on some clinical isolates, no significant correlation was shown among the enterotoxigenic strains and coagulase gene (Demir et al., 2011 ▶). Similar results were obtained by Rojasandda-Cunha Mde L. (Da et al., 2007 ▶; Suarez et al., 2008 ▶; Rojas et al., 2012 ▶). Although the coagulase and nuclease tests are valuable to identify S. aureus, the tests do not necessarily imply entero-toxigenic strains.

It could be concluded from the study, the sensitivity and reliability of the PCR assay detects the staphylococcal enterotoxin. Additionally, the presence of the genes was associated with the enterotoxigenic bacteria and therefore, to detect the enterotoxigenic types of S. aureus, genotypic methods are highly re-commended. Also, no correlation was shown between the production of enterotoxin, and presence of coagulase and thermonuclease genes.

Acknowledgment

The work was supported by School of Veterinary Medicine, Shiraz University, Iran.

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