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05-Pages: 01                                           2020-09-2020 | Published: 19-08-2020 | Accepted: 10-07-Received: 30 

                                                                                                                                                                                           

Clinical Medicine Insights Page 1 
 

 

Proliferation of Tst Genes in Methicillin-Resistant Staphylococcus aureus 

(MRSA) 

Ihsan A. Alkhafaji 

Microbiology Department, College of Medicine Iran 

Abstract : Methicillin-resistant Staphylococcus aureus (MRSA) is a common and important cause of 

nosocomial infections not found in hospitals and health care facilities where people are poorly protected, 

MRSA is commonly spread in hospitals by patients as a primary reserve, health care workers (HCWs), 

surrounding environments, and sometimes airborne, MRSA possesses a number of toxicity factors that play 

an important role in the spread and resistance of bacteria, including a super-antigen encoded by the Tst gene 

Toxic Shock Syndrome-1 ( TSS-1) Infectious complications are secondary forms of invasive bacterial 

diseases such as pneumonia, lung disorders, urinary tract infections, food poisoning, arthritis, endocarditis, 

meningitis, arthritis, toxic shock syndrome, sepsis and m. The study found the presence of methicillin-

resistant staphylococcal bacteria (MRSA), 39/46 (85%), the Tst gene was absent in most isolates, only 6/39 

(15.4%) of the Tst gene. 

Key words: Staphylococcus aureus (MRSA), Genê Tst 

Introduction 

Staph aureus is one of the most common pathogenic bacteria, as its pathology is dependent on a group of 

virulence factors that affect the host and cause the disease. (Hoseini Alfatemi et al. 2014)It is also one of the 

"ESKAPE" organisms, can cause many serious infections so it is considered a serious and growing threat 

worldwide that can affect various groups and can cause serious nosocomial infections. (Liang et al. 2019) 

Staph aureus can avoids further clearance by the immunity through the expression of surface-linked proteins 

and polysaccharide capsules that prevent opsono-phagocytic killing. Staphylococcus protein A (SpA) is a 

membrane-bound protein that binds with Fc region in IgG, thereby avoiding recognition of macrophages 

(Walton 2013) The success of Staph aureus a pathogen is explained by it ability to express: 

 (1) Its ability to invade and inflammate. This includes a number of mechanisms including colonization, 

synthesis of extracellular structure of molecules that facilitate adherence and help them avoid host defenses. 

 (2) Its ability to produce toxins. (Zhu 2010) and(Haghkhah 2003) 

 Neutrophils are the decisive defense of the body in controlling the colonization and spread of Staph aureus 

despite the behavior of Staphylococcus for many evasion strategies.One of these strategies is the induction 

of neutrophil cell death, which causes inflammations and tissue damage and increased disease severity. 

(Yang et al. 2019) In addition to evading strategies, Staph aureus produces many virulence factors, which 

include enzymes and toxins, in addition to its ability to produce septic shock by activating and interacting 

with the immune system and coagulation (Rigby and DeLeo 2012) and  (Gordon and Lowy 2008) .   Staph 

aureus produces a wide variety of exotoxins, among the numerous toxins of including enterotoxins, the 

enterotoxins super antigens have already been assigned to the pyrogenic toxin super antigen family based on 

their biological activity and structural similarity, toxic shock toxin-1 (TSST-1) that induces super antigenic 

activity, and exfoliative toxins (ETs), these toxins are responsible for specific acute clinical syndromes such 

as toxic shock syndromes (TSS) , food poisoning due to staphylococcus enterotoxins and staphylococcal 

scarlet fever (a mild form of TSS), all these toxins share in their structural and biological properties, and this 

indicates that they are derived from a common ancestor. (Zhu 2010) and (Thomas et al. 2006) . Another 

class of genetic characteristics of staphylococci is a super-antigen that encoded by Tst gene, that carried on 



Proliferation of Tst Genes in Methicillin-Resistant Staphylococcus aureus (MRSA 

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mobile genetic elements (MGE) named (SaPIs.), nearly 15 kb genomic regions that significantly denote a 

number of virulence genes, (SaPIs)  linked to specific Staph aureus genetic families, known as lineages 

(Sharma et al. 2018) and (Shien 2014) Toxic shock syndrome-1 (TSS-1) Secondary inflammatory 

complications include invasive forms of bacterial diseases Such as inflammation of the lungs, lung 

abscesses, urinary tract infections, food poisoning, osteoarthritis, Endocarditis, meningitis, arthritis, toxic 

shock syndrome, septicemia, Death (Bocskay 2016)  

Materials and methods 

Sampling 

A total of 484 samples collected from patients (Skin swab, Nasal swab and Wound swab), Health Care 

Workers (Skin swab and Nasal swab), hospital words (Orthopedic and Surgical words) and Operation 

Theater (various places of Operation Theater before and after sterilization). Samples were collected in the 

period Between November 2018 and August 2019, from two locations, Al-Basrah Teaching Hospital and 

Al-Saddr Teaching   Hospital.  Each swab was transferred in to enrichment medium (brain heart infusion 

broth (BHIB)) for 2-4 hour at 37
o
C. (Nicholas P. Vitko and Anthony R. Richardson 2014)  

Culture and identity 

Staphylococcus grows easily on most routine media at aerobic or micro-aerophilic conditions. It was quickly 

grows at (37°C), and  the ideal  temperature in which the pigment is formed is 20-25°C Staph aureus usually 

forms grey to golden yellow colonies due to carotenoids, Produces β-haemolysis on horse, sheep or human 

blood agar plates (Suzuki et al. 2012) and (Gillet et al. 2002) The bacterial morphology was observed 

microscopically as Gram-positive cocci arranged in grape-like irregular clusters (Gillet et al. 2002) All 

Staph aureus strains produce coagulase enzyme. Staph aureus are catalase positive and oxidase negative 

(Suzuki et al. 2012) Staph aureus express a clumping factor (fibrinogen affinity factor) (Reddy, Srirama, and 

Dirisala 2017) Staphylococcus can grow in a medium with a high salt concentration, so they can grow easily 

in MSA. The acidity of the medium changes as the bacteria ferments mannitol and turn phenol red pH-

indicator; Staph aureus changes color of MSA from the alkaline (red)to the acidic(yellow), while the rest of 

the Staphylococcus will grow without changing the color of the medium. (Gillet et al. 2002) 

Detection of MRSA 

• Cefoxitin disc diffusion, Significant method to detect MRSA, by testing MRSA resistance to the 

cefoxitin disc, culture was done on MHA plate , incubation temperatures at 35-36 
0
C and times of 18-24 

hour  strains of Staph aureus having zone of inhibition less than 19mm defined as MRSA. (Brown et al. 

2005)  

•   PCR Methods, Strains of Staph aureus harboring MecA gene defined as MRSA (Brown et al. 2005) 

Genomic DNA extraction 

As the instruction of manufacturer (promega company) Polymerase Chain Reaction (PCR) according to the 

manufacturer's instructions, the DNA were detected by gel electrophoresis, the samples were loaded in 0.8% 

agarose gel 1×TBE (54 g Tris-base, 0.5M EDTA, 1-l distilled water, pH=8 and diluted with 400 ml of 

distilled water) and electrophoresed at 60 V for 30 min. 

Polymerase chain reaction technique  

PCR is a very effective method to amplify a particular DNA as many copies of a specific DNA (Bartlett 

2003), all MRSA isolates were assayed for the presence of the Tst gene by PCR using previously described 

primers, for PCR used diluted forward and reverse primers to reach (100 pmol/μl) concentration as stock 

solution, distilled water was used as the negative control. 

Statistical analysis 

Statistical analysis was done using SPSS (Statistical Package for Social Sience) program V. 20, 

Experimental data were presented in terms of observed numbers and percentage frequencies, and then 

analyzed by using Chi-square (χ2) test to determine the relationship between the variables, P value ≤ 0.05 



Proliferation of Tst Genes in Methicillin-Resistant Staphylococcus aureus (MRSA 

Clinical Medicine Insights Page 3 
 

was considered statistically significant.  

Results  

Identification of bacterial isolates 

Samples were collected in the period between November 2018 and August 2019 from two locations, Al-

Basrah Teaching Hospital and Al-Sadder Teaching Hospital. Out of 485 samples only 46 (9.48 %) were 

identified as coagulase positive staphylococci, as shown in table (3-1) PCR product was electrophoresed in 

1.5 % agarose gel, Stained with ethidium bromide, ,  7 μL of PCR products and promega DNA ladder (50-

1000bp)  carefully loaded in the wells and electric current was matched (65 volt for 45 h). The gel was then 

observed under a UV light and compare with ladder (50-1000bp). 

Detection of MRSA isolate 

Cefoxitin resistance staph aureus isolate harboring mecA gene (MRSA) was detected in 39from 46(85%) S. 

aureus isolates. 10(90%) isolates of MRSA were from wound samples, 6 (75%) from patient skin swab and 

5 (83%) from patient noses, 4(80%) from hospital wards, 4(80%) from health care workers hands, 7(100%) 

from health care workers noses and 3 (75%) from hospitals theaters samples. 

MecA gene detactions 

Staphylococcus MecA gene presence in all MRSA  isolates,   to detect Staph aureus isolates with MecA 

gene, it was subjected to PCR technique, MecA gene band detected at 147bp region. 

Tst gene detection 

Tst gene detected only in 15.4% of MRSA isolate. 

After amplification by PCR technique.  Genes were detected by Gel electrophoresis of amplified PCR 

products of Tst genes (326bp) of Staph aureus isolates in PCR techniqError! Reference source not found. 

Sequencing for Tst  gene 

Query isolate (our isolate) begin from (9-291) bp when compared with subject isolate (Stander isolate) begin 

from (455638-455356) bp, where the compatibility occur between the two isolates for identification Query 

isolate, identities was 98%, There was a mismatch at seven places. 

Discussion 

The study showed that these bacteria isolated from the hospital environment (operating rooms and patients' 

rooms) and hands and noses of workers and patients may be causes wound infection, There is increasing 

concern about MRSA contamination and infections in the hospital words meanly in post-operative wound, 

in our study isolation showed high prevalence range of MRSA strains 85% (39/46) of the total staph aureus 

isolated from various samples, higher rate of MRSA isolations from H.C.W nasal swabs 100% (7/7), 

followed by wound swabs 90.9% (10/11), Nasal swab from patient 83% (5/6), H.C.Ws hand swabs and 

hospital words 80% (4/5) for each, lowest rate were recorded for the patient Skin swab (6/8) and operative 

rooms (3/4) 75% each. The finding about high prevelance of MRSA is not surprising and is also in line with 

several studies carried out in Iraq. (Al-azawi et al. 2016) ; (Al-dahbi and Al-mathkhury 2013) and (Al-

Maliki 2009) The ratio of MRSA was relatively low in a study conducted in Kurdistan region of Iraq, in 

2015 where the MRSA prevalence was 53% (Hussein et al. 2015) In another study in Iran was 69% 

(Jahanshahi, Zeighami, and Haghi 2018) while in a study conducted in India, the percentage was much 

lower 16.6% (Goud et al. 2011) MRSA prevalence 51.4% at the Korean hospital from the Staph aureus 

collected from blood and nasal colonizers (Peck et al. 2009) In general MRSA was highly prevalent in Asian 

countries (Hussain et al. 2019) In the German study there was a decrease in MRSA rate (Schubert, Kämpf, 

Wahl, et al., 2019b) In Turkey 2017, high rates of staph aureus high resiste to penicillin and ampicillin 

(Yılmaz and Aslantaş 2017) A study in Isfahan, Iran, in 2018 showed a nasal carriage of MRSA 51.9% 

among patient and 16% among health workers (Moshtagheian et al. 2018) MRSA prevalence in wounds was 

high, and this is consistent with previous studies, 76.9% (Khanal and Jha 2010); 44% (Tyagi, Kapil, and 

Singh 2008); 60.1% (Orrett and Land 2006); 34.8% (Hafeez, Chughtai, and Aslam 2004)  The presence of 

MRSA in wounds delays healing (Solomkin 2001). In other study the average of MRSA rate for Wound 

Infections After Cardiothoracic Surgery was 54% in a three-year period from 2007 to 2010 (Walsh, Greene, 



Proliferation of Tst Genes in Methicillin-Resistant Staphylococcus aureus (MRSA 

Clinical Medicine Insights Page 4 
 

and Kirshner 2011) During the present study, MRSA isolate from HCWs noses was (19.4%) Which 

corresponds to previous studies (Caceres 2011); (Shittu et al. 2011) and (Munoz et al. 2008); 12.7% 

(Shibabaw, Abebe, and Mihret 2013) 12% in (Ibarra et al. 2008); 14.3% (Radhakrishna et al. 2013),  MRSA 

rate was low in other studies; only 5.3% in Iran study (Askarian et al. 2009); and it was 0% in Kenya 

(Omuse, Kariuki, and Revathi 2012) With increasing of MRSA colonization rate, there is greater risk in 

developing drug-resistant wound infections. Therefore, it is necessary to avoid infection as much as 

possible. Previous reserchs have shown a large infections rate due to cross-contamination by hands of health 

staff (Rotter and Koller 1991) All MRSA isolate in our study harboring MecA gene. This result agreed with 

many other studies that showed all MRSA isolate harboring MecA gene (Yang et al. 2020); (Mussa and Al-

mathkhury 2018); (Karmakar, Dua, and Ghosh 2016)(Al-Charrakh, Al-Hassnawi, and Al-Khafaji 2015);  

(Dağı et al. 2015) Other studies considered that Methicillin resistance can be happen in MecA absence, 

MRSA could have other mechanism(s) for resistance; e.g., altered target site or may be reduced drug 

accumulation. MecA gene absence may also due to a technical error upon detection. (Mahdi et al. 2016); 

(Carpaij et al. 2011) and (Wielders et al. 2002) The TSST coded by Tst gene (Dinges, Orwin, and Schlievert 

2000) The Tst gene was detected in (15.4%) MRSA isolates in our study, The percentage was close to these 

studies (Ezeamagu et al. 2018) and (Hoseini Alfatemi et al. 2014) (14%,11.6%) respectively.In other studies, 

the Tst gene ratio was slightly higher, Tst gene was detected in (26.31%) (Costa et al. 2018) (27.9%) 

(Megevand,et,al,2010), Other studies recorded highly prevalent Tst gene (72.2%) in MRSA isolates from 

blood (Peck et al. 2009) Whereas in another study, Tst genes were non-detected (Motallebi et al. 2019) 

 

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