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ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 265-268
Prevalence of enterotoxin a and b genes in Staphylococcus aureus isolated from clinical samples and healthy carriers in Gorgan City, North of Iran


1 Department of Microbiology and Infectious Disease Research Center, Golestan University of Medical Science, Gorgan, Iran
2 Department of Biology, Azad University Gorgan Branch, Iran

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Date of Web Publication24-Oct-2013
 

   Abstract 

Background: Infections due to Staphylococcus aureus, a nosocomial and community-acquired pathogen, is a major public health problem. Wide range of diseases caused by S. aureus from mild infections of the skin and soft tissue to life threatening diseases which is due to having several virulence factors such as enzymes, toxins and also enterotoxins. Enterotoxin A (SEA) and enterotoxin B (SEB) are superantigens and gasterointestinal toxins causing food poisoning. The sea and seb genes encode SEA and SEB, respectively. The goal of this study was determine the prevalence of sea and seb genes in S. aureus isolated from patients and healthy carriers in Gorgan city, north of Iran. Materials and Methods: 170 isolates of S. aureus (95 from patients and 75 healthy carriers) were collected during 1 year. After identification and purification, DNA extraction was done by phenol - chloroform method. Amplification of sea and seb genes was done by specific primers and polymerase chain reaction method. Results: Among the 170 isolates of S. aureus, 60.6% and 27.1% contained sea and seb genes, respectively. The frequencies of isolates containing sea and seb genes were 58.8% and 61.3%, respectively, in methicillin-resistant Staphylococcus aureus (MRSA) isolates and 23.5% and 28.6%, respectively, in methicillin-sensitive Staphylococcus aureus (MSSA) isolates which were not statistically significant. The frequency of these genes was not related to age, sex and source of isolation in the patients. Conclusion: This study showed that a high proportion of S. aureus isolates carried sea gene, whereas the frequency of seb gene in this region was predictable.

Keywords: Enterotoxin A, Enterotoxin B, polymerase chain reaction, Staphylococcus aureus

How to cite this article:
Kamarehei F, Ghaemi EA, Dadgar T. Prevalence of enterotoxin a and b genes in Staphylococcus aureus isolated from clinical samples and healthy carriers in Gorgan City, North of Iran . Indian J Pathol Microbiol 2013;56:265-8

How to cite this URL:
Kamarehei F, Ghaemi EA, Dadgar T. Prevalence of enterotoxin a and b genes in Staphylococcus aureus isolated from clinical samples and healthy carriers in Gorgan City, North of Iran . Indian J Pathol Microbiol [serial online] 2013 [cited 2019 Jan 20];56:265-8. Available from: http://www.ijpmonline.org/text.asp?2013/56/3/265/120388



   Introduction Top


Staphylococcus aureus is a nosocomial and community-acquired pathogen that can cause serious infections. [1] The pathogenesis of S. aureus is caused by several virulence factors such as staphylococcal exotoxins. [2] S. aureus enterotoxins (SEs) belong to a large family of pyrogenic exotoxins and they have common genetic relationships, structure, function and sequence homology. These toxins can cause food poisoning and several allergic and autoimmune diseases. SEs are heat-stable enterotoxins that have A-V (except F) serological types. [3] They are potent gastrointestinal toxins and superantigens that stimulate non-specific T-cell proliferation. Although SEA and SEB are localized on separate domains of the proteins, there is a high correlation between their activities and superantigen activity. [4]

The most common staphylococcal enterotoxins (SEs) are SEA and SEB. SEA is the most common toxin in Staphylococcus-related food poisoning. While SEB is also associated with food poisoning, it has been studied as an inhaled bioweapon. [5] The goal of this study was to determine the prevalence of sea and seb genes in S. aureus isolated from patients and healthy carriers in Gorgan city, north of Iran.


   Material and Methods Top


Bacterial isolates

In this research, we studied 170 isolates of S. aureus (95 from patients and 75 healthy carriers) that were collected during 1 year. Purification and confirmation of diagnosis of S. aureus by biochemical tests were carried out as previously mentioned. [6],[7],[8]

DNA extraction

DNA was extracted using phenol-chloroform method that used lyzozyme, sarcosyl, proteinase K, RNase, phenol and chloroform, without using lysostaphin and boiling. [9] The purified DNA was assessed by electrophoresis and stored at -20°C.

Sea and seb gene detection

Polymerase chain reaction (PCR) was carried out for all the isolates (Eppendorf Thermocycler, Hamburg, Germany) for detecting sea and seb genes in a final volume of 25 μl. The primers designed by Imani-fouladi (unpublished data) were used [Table 1]. PCR matrix contained 0.5 μl of Taq polymerase (GENET BIO-Korea), 1 μl of the DNA template, 3 μl MgCl 2 (GENET BIO), 1 μl of deoxyribonucleotide triphospate dNTP (GENET BIO-Korea), 1 μl of each primer and 17.5 μl of DNA free water. DNA isolates were denatured for 5 min at 94°C, followed by 35 cycles of denaturing performed for 60 s at 94°C, with annealing at 50°C for sea and at 55°C for seb for 60 s and extension at 72°C for 60 s. Finally, 5 min of final extension was performed at 72°C. PCR products were analyzed by electrophoresis through 2% agarose gel with ethidium bromide.

Methicillin susceptibility testing

Susceptibility to methicillin was determined by the agar disk diffusion method using Muller Hinton agar medium containing 2% NaCl with oxacillin disk (MAST Diagnostics, Merseyside, UK). All plates were incubated at 35°C overnight. Molecular detection of this gene was performed. [7]

The data were entered in SPSS version 16 and χ2 test was used for statistical analysis. P-value of less than 0.05 was considered to be statistically significant.


   Results Top


Among the 170 S. aureus isolates, 103 (60.6%) cases were carrying sea gene. The distribution of these isolates among the people aged less than 20 years (62.5%) and in healthy carriers (65.3%) was higher than in other groups. The frequency of sea gene among MSSA (Methicillin-sensitive Staphylococcus aureus) isolates (61.3%) was more than in MRSA (Methicillin-resistant Staphylococcus aureus ) isolates [Table 2], but there was not any significant difference between them (P > 0.05).

Also, 46 (27.1%) of the tested S. aureus isolates contained seb gene. Most of the seb-positive isolates were found in women (28.7%), age group 20-45 years (30.6%), healthy carriers (32%) and MSSA (28.6%) [Table 3]. There was not any statistical difference between the different studied groups (P > 0.05) .
Table 1: Primers used for sea and seb genes for detection in S. aureus isolated in north of Iran

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Table 2: Characteristics of patients and healthy carriers of Staphylococcus aureus enterotoxin a-producing gene

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Table 3: Characteristics of patients and healthy carriers of Staphylococcus aureus enterotoxin b-producing gene

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   Discussion Top


Staphylococcal food poisoning is one of the major concerns as a public health problem. To date, around 250 different food-borne diseases have been described. Contaminated food with S. aureus can causes gastroenteritis in human. [10 ]

SEs produced by S. aureus belong to the fascinating family of superantigens, which repress the immune system (innate and specific immune responses) of the host. [11] Studies in the US were showed a high frequency of sea-carrying strains (up to77.8%). [4] On the other hand, while seb is associated with food poisoning, it has been studied as an inhaled bioweapon. [12]

Our study demonstrated that 60.6% of S. aureus isolates were sea positive. It is interesting to note that this prevalence is higher than that reported in some other studies performed in various regions in Iran, such as Tehran, [13] as well as in some other countries. [14],[15],[16] It may be due to the presence of a specific strain of S. aureus carrying sea gene in our region, which needs to be studied further.

In the present study, the frequency of sea-positive isolates observed in blood specimens was 72.7%, which may be related to its role in virulence. For this reason, further study on the expression of sea gene is proposed. Also, we found that sea gene has been detected in MSSA more than in MRSA isolates, which is compatible with the previous observations [14],[16],[17] that confirmed an inverse relationship between sea and mecA genes.

Another point worth mentioning is that the sea gene was much frequent in S. aureus isolated from healthy carriers, men and people of age less than 20 years than in the other tested groups, but this difference was not statistically significant (P > 0.05).

Our study measured the frequency of sea gene and the reason for the high frequency of this gene in healthy carriers than in patients is unknown. It may be because S. aureus carrying enterotoxins are transferred easily between healthy carriers than patients and can cause infections in their host. Indeed, it is our question and further studies with animal models need to be done to determine how the presence of Staphylococcal toxins can influence the pathogenicity of S. aureus infections.

Frequency of seb gene among S. aureus isolates in our region was 27.1%, which is similar to some other studies performed in various regions which showed 27.4% and 23% frequencies. [14],[16] But in many other studies, the frequency of this gene was reported to be lesser than our finding (8%, 7.4% and 6%). [17],[18],[19] This may be related to the different epidemiological factors such as type of strain, type of specimen and condition of the patient.

Most of the seb-positive isolates were found in women, age group 20-45 years, healthy carriers, MSSA and isolates from urine than in the other tested groups. But this difference was not statistically significant (P > 0.05).

In our study, notably, 32 (18.8%) of S. aureus isolates contained both sea and seb genes. Moreover, among 124 seb-negative isolates, there were 53 sea-negative isolates; in other words, when seb is negative by PCR, 79.1% sea is negative too.

Based on our findings, typing of S. aureus and sea gene expression among different strains present in this region for detection and control of strains contain sea gene are proposed.


   Acknowledgments Top


We would like to acknowledge A. Imani Fouladi for the designing of sea, seb primers and Infectious Disease Research Center of Golestan University of Medical Sciences for the financial support.

 
   References Top

1.Fey PD, Said-Salim B, Rupp ME, Hinrichs SH, Boxrud DJ, Davis CC, et al. Comparative molecular analysis of community- or hospital-acquired methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2003;47:196-203.  Back to cited text no. 1
    
2.Gravet A, Colin DA, Keller D, Girardot R, Monteil H, Prevost G. Characterization of a novel structural member, LukE-LukD, of the bi-component staphylococcal leucotoxins family. FEBS Lett 1998;436:202-8.  Back to cited text no. 2
    
3.Schelin J, Wallin-Carlquist N, Cohn MT, Lindqvist R, Barker GC, Radstrom P. The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment. Virulence 2011;2:580-92.  Back to cited text no. 3
    
4.Balaban N, Rasooly A. Staphylococcal enterotoxins. Int J Food Microbiol 2000;61:1-10.  Back to cited text no. 4
    
5.Pinchuk IV, Beswick EJ, Reyes VE. Staphylococcal enterotoxins. Toxins 2010;2:2177-97.  Back to cited text no. 5
    
6.Rahimi-Alang S, Cheraghali F, Yazarlou S, Amini A, Shakeri F, Ghaemi E. Frequency of methicillin resistant Staphylococcus aureus in health care. Zahedan J Res Med Sci 2011;13:17-22.  Back to cited text no. 6
    
7.Vaez H, Moradi A, Ghaemi EA. Evaluation of methicillin resistance Staphylococcus aureus isolated from patients in Golestan province-north of Iran. Afr J Microbiol Res 2011;5:432-6.  Back to cited text no. 7
    
8.Rastegar Lari AA, Ohadian Moghadam S, Abdossamadi Z, Asghari B. Prevalence of PVL-containing MRSA isolates among hospital staff nasal carriers. Lab Med 2011;42:283-6  Back to cited text no. 8
    
9.Shakeri F, Golalipour M, Rahimi Alang S, Ghaemi EA. Spa Diversity among MRSA and MSSA strains of staphylococcus aureus in North of Iran. Int J Microbiol 2010;2010. pii: 351397.  Back to cited text no. 9
    
10.Le Loir Y, Baron F, Gautier M. Staphylococcus aureus and food poisoning. Genet Mol Res 2003;2:63-76.  Back to cited text no. 10
    
11.Argudin MA, Mendoza M, Rodicio MR. Food Poisoning and Staphylococcus aureus enterotoxins. Toxins (Basel) 2010;2:1751-73.  Back to cited text no. 11
    
12.Ahanotu E, Ravita T, Gaunt E. Staphylococcal enterotoxin B as a biological weapon: Recognition, management and surveillance of staphylococcal enterotoxin. Appl Biosaf 2006;11:120-6.  Back to cited text no. 12
    
13.Pourmand MR, Bagherzadeh Yazdchi S. High prevalence of SEA gene among clinical isolates of staphylococcus aureus in Tehran. Acta Med Iran 2009;47:354-61.  Back to cited text no. 13
    
14.Jimenez JN, Ocampo AM, Vanegas JM, Rodriguez EA, Garces CG, Patino LA, et al. Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellin, Colombia. Mem Inst Oswaldo Cruz 2011;106:980-5.  Back to cited text no. 14
    
15.Nada HA, Gomaa NI, Elakhras A, Wasfy R, Baker RA. Skin colonization by superantigen-producing Staphylococcus aureus in Egyptian patients with atopic dermatitis and its relation to disease severity and serum interleukin-4 level. Int J Infect Dis 2012;16:e29-33.  Back to cited text no. 15
    
16.Barretti P, Moraes TM, Camargo CH, Caramori JC, Mondelli AL, Montelli AC, et al. Peritoneal dialysis-related peritonitis due to Staphylococcus aureus: A single-center experience over 15 years. PLoS One 2012;7:e31780.  Back to cited text no. 16
    
17.Fouladi AI, Mehrabadi JF. Study of prevalence of Enterotoxin type B gene in Meticillin Resistant Staphylococcus aureus (MRSA) isolated from wound. Kowsar Med J Spring 2011;16:21-5.  Back to cited text no. 17
    
18.Souza RR, Coelho LR, Botelho AM, Ribeiro A, Rito PN, Vieira VV, et al. Biofilm formation and prevalence of lukF-pv, seb, sec and tst genes among hospital- and community-acquired isolates of some international methicillin-resistant Staphylococcus aureus lineages. Clin Microbiol Infect 2009;15:203-7.  Back to cited text no. 18
    
19.Wang L, Safo M, Archer GL. Characterization of DNA sequences required for the CcrAB-mediated integration of staphylococcal cassette chromosome mec, a Staphylococcus aureus genomic island. J Bacteriol 2012;194:486-98.  Back to cited text no. 19
    

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Correspondence Address:
Ezzat Allah Ghaemi
Department of Microbiology and Infectious Disease Research Center, Golestan University of Medical Science, Gorgan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.120388

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    Tables

  [Table 1], [Table 2], [Table 3]

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