Polymorphism Analysis of the CoA (Coagulase) Gene in Isolates of Methicillin-Resistant Staphylococcus Aureus with AluI Restriction Sites

Analysis of the polymorphism of a gene is important to obtain early information in identifying genetic markers related to the characteristics to be seen. The RFLP method becomes one of the chosen methods because it can see polymorphism that can be detected by using the different fragments of DNA that have been cut by using certain endonuclease enzyme so that it is possible to describe the polymorphism of a gene. Application of RFLP in this study was devoted to see the coagulase gene polymorphism of methichillin-resistant S. aureus. Based on the description above, researcher was interested in conducting the analysis of CoA (coagulase) gene polymorphism with AluI restriction site of methicillin-resistant S. aureus isolates. The results of this study are aimed to be scientific information about the genetic variation of the coagulase gene of methicillin-resistant S. aureus, and as information in the management of diseases related to antibiotic resistance. This was a descriptive study intended to discover the coagulase gene polymorphism of methichillin-resistant S. aureus. The research was conducted in Microbiology Laboratory of Health Analyst Department, Surabaya, and Human Genetic Laboratory of Institute of Tropical Disease of Airlangga University Surabaya. Samples from this study was a collection of isolates of MRSA in Microbiology Laboratory of Dr. Soetomo Hospital Surabaya selected by using consecutive sampling. Based on the bacteriological examination, all of the samples were S. aureus and the results of amplification showed that PCR product (amplicon) of mecA and CoA gene from specific primers, that were 304 bp and 756 bp, had a positivity of 100%. The results of PCR-RFLP of CoA gene showed that all 25 samples underwent polymorphism which was divided into four RFLP patterns and the biggest one was RFLP I pattern (the one which was not digested by AluI restriction enzyme) with a proportion of 64%.


Methicillin-Resistant Staphylococcus aureus (MRSA)
is a Staphylococcus aureus that become insusceptible or resistant by methicillin antibiotic types. 1 MRSA become resistant because of genetic changes that caused by exposure of irrational antibiotic therapy. 1 Patients with S. aureus colonization are the major transmission sources of S. aureus at hospitals and are also responsible for the clinical infections of other patients. 2 The prevalence of MRSA in various hospitals in the world ranges from 2-70% with an average of 20%. 3 In recent decades there is an increased prevalence of S. aureus and MRSA in the world. A study based on the population of North America and Europe indicated that the prevalence of S. aureus was between 18-30%. 4 In overall Asia, MRSA prevalence has reached 70% 5 , while MRSA publication and prevalence in Indonesia are still very limited and difficult to obtain. S. aureus is able to produce coagulase, an enzyme protein which is capable of coagulating plasma oxalate or plasma citrate. Bacteria producing coagulases are considered having the potential to become invasive pathogens. A coagulase enables S. aureus to avoid phagocytosis through the formation of protective wall layers in the forms of fibrin. S. aureus pathogenicity is due to the toxin production working after bacteria successfully enter and survive in the hosts' body. In this initial phase, a coagulase serves as a virulence factor which is capable of coagulating proteins by protecting bacteria from phagocytosis and inhibiting the antibiotic penetration. As the treatment is difficult to perform, those bacteria may cause infection and antibiotic resistance. 8 A gene polymorphism analysis is necessary to conduct to obtain earlier information in identifying genetic markers related to the desirable characteristics to see. Restriction fragment length polymorphism (RFLP) is a method selected to see the DNA sequence homologues which may be detected using DNA fragment differences cut using a certain endonuclease enzyme which is capable of describing a gene polymorphism. The analytical studies on methicillinresistant S. aureus coagulase gene polymorphism using AluI restriction enzyme have been conducted on methicillin-resistant S. aureus isolate population in Iran, India, and Europe, which result in different RFLP patterns. [8][9][10][11] In Indonesia, studies on clinicalisolates of methicillin-resistant S. aureus coagulase gene polymorphism with AluI restriction enzyme have never been conducted before that the researcher is interested in conducting polymorphism analysis of the coagulase gene in clinical isolates of methicillinresistant S. aureus with AluI restriction sites by RFLP. The application of RFLP in this research is especially used to see the presence of methicillin-resistant S. aureus polymorphism coagulases. Based on above descriptions, the researcher is interested in conducting analysis on coagulase gene with AluI restrictive site of methicillin-resistant S. aureusisolates.

Bacterial Isolates
The samples (25) were collected from patients suspected for MRSA infections in RSUD DR. Soetomo Surabaya. In addition one strains of Staphylococcus aureus subsp 6850 were maintained as positive control for identification of coagulase gene polymorphism.
The collected samples were streaked on blood agar medium and incubated at 37 o C for about 24 hours.

Identification of S.aureus
Several tests were carried out for the identification of S. aureus. Initially Gram's staining was performed to identify the morphology of the organism, and various biochemical tests such as catalase test, oxidase test, mannitol salt agar test, slide coagulase, motility, citrate, and fermentation of carbohydrates (fructose, galactose, lactose, maltose, mannose, sucrose) test were carried out.

DNA extraction
Genomic DNA from the bacteria was extracted from the samples with chloroform-phenol method. Initially 1.5 ml bacterial culture was taken and subjected to centrifugation. The resulting pellet was add with 1000 μl of DNAzol and itwas incubated at 25°C for 20 minutes, after that add 200 μl of chloroform, vortex. Then the suspension was centrifuged. Later 600 μl of isopropanol was added and centrifuged. The pellet was washed with 75% ethanol and dried. Finally, the DNA pellet was stored in nuclease free water. Then the DNA fragments were measured by Nanodrop ™ 2000 Thermo Scientific spectrophotometer.

Identification of mecA gene methicillin-resistant in S. aureus by PCR
The mecAgene was amplified by using two sequence of the primers by using forward primer: 5′ TGGCTATCGTGTCACAATCG 3′ and reverse primer: 5′ CTGGAACTTGTTGAGCAGAG 3′. 3 S. aureus isolates were tested for the presence of the 304-bp PCR product of the mecA gene. The initial denaturation was at 94°C for 30 second. Denaturation of 94°C for 1 minute, annealing at 52°C for 30 second and elongation temperature of 72°C for 1 minute was maintained for 30 cycles. The final elongation was at 72°C for 5 minutes and the reaction was held at 4°C. The amplified products were subjected to 2% agarose gel electrophoresis.

Identification of coagulase gene methicillinresistant in S. aureus by PCR
The coagulase gene was amplified by using two sequence of the primersby using forward primer: 5′ ATAGAGATGCTGGTACAGG 3′ and reverse primer: 5′ C GCTTCCGATTGTTCGATGC 3′. 11 S. aureus isolates were tested for the presence of the 756-bp PCR product of the coagulase gene. The initial denaturation was at 94°C for 45 second. Denaturation of 94°C for 20 minute, annealing at 57°C for 30 second and elongation temperature of 72°C for 30 minute was maintained for 30 cycles. The final elongation was at 72°C for 3 minutes and the reaction was held at 4°C. The amplified products were subjected to 2% agarose gel electrophoresis.

PCR-RFLP of coagulase gene S. aureus
The RFLP of coagulase gene was carried out by using AluI, 10μl of coagulase gene PCR products were digested with 2U AluI restriction enzyme according to the manufacturer's instructions (AluI Thermo Scientific) and incubated at 37ºC for 3 hour. The resulting restricted fragments were separated in polyacrylamide gel.

Isolation and identification of S. aureus
Twenty five different samples were collected from clinical patients. Among these samples, 25 samples resulted in individual spherical colonies of greyish white color. The isolates with individual spherical, greyish white colonies were again sub cultured in a new blood agar medium and were incubatedat 37 o C for 24 hours. The results of bacteriological examination are conducted using a conventional method. 12 The following results were obtained (Figure 1), it can be concluded that all of the samples are S. aureus.

Identification of coagulase gene methicillin-resistant in S. aureus by PCR
Identification of coagulase gene methicillin resistance in S. aureus was performed by PCR. The results were observed under 2% agarose gel electrophoresis with 100-1500 bp DNA marker. The following results were obtained (Figure 3), it can be concluded that 100% samples are positive coagulase gene.

PCR-RFLP of coagulase gene S. aureus methicillin resistant (MRSA)
The PCR amplicons were digested with AluI restriction enzyme and five different restriction patterns were obtained ( Figure 4) ( Table 1). This difference in restriction pattern may be due to the polymorphism existing in the coagulase gene of different MRSA isolates. There are several variation differences of RFLP MRSA patterns in coagulase gene found in this study when compared with those found by the other researchers which shows that the strains of methicillin-resistant is highly S. aureus polymorphic shown by the sequential changes of AluI enzyme introduction in coagulase gene since there is a point mutation. These sequential changes may cause genetic diversity of S. aureus coagulase genes. The presence of genetic diversity in coagulase gene is in accordance with the results of research that the coagulase gene polymorphic is also due to the differences in sequence 3' of coagulase gene regional variable on S. aureus strains which may result in various amino acid sequential encoding in coagulase gene. 8 It is predicted that this coagulase gene polymorphism has an important role in antibiotic resistance occurring in S. aureus bacteria. Coagulase enzyme has the function to coagulate plasma oxalate or plasma citrate as there is a reactive coagulase factor in the serum which reacts with the enzyme. The esterase resulted may increase the coagulation activity that fibrin deposit is formed on the surface of bacterial cells. Fibrin deposit on the surface of bacterial cells may cover pores of the bacterial cell walls which inhibit the antibiotic penetration as to many antibiotics enter the bacterial cells through the pores of the cell walls and membranes. The antibiotic concentration in cells is equal with the number of pores in the cells that bacteria resistant to antibiotics have the mechanism to reduce or close the pores on cell membranes by maximizing the activities of coagulase enzymes to inhibit the antibiotic absorptions and enable the number of antibiotics entering the cell is inadequate to damage or kill the bacteria. 14   In addition, S. aureus antibiotic resistance is also caused by the inappropriate use of antibiotics. Thus, antibiotic abuses are the common causes of antibiotic resistance, including the first antibiotic uses for viral infections and less optimized antibiotic dosage uses may result in bacterial antibiotic resistance. 15,16 Analysis on various virulence and resistance factors which are responsible for S. aureus pathogenicity is essential to conduct. This research emphasizes on the importance of methicillin-resistant S. aureus positive coagulase molecular diagnosis. Polymorphism analysis in coagulase gene is extremely helpful in treating various infections caused by methicillin-resistant S. aureus based on its genotype.

DISCUSSIONS
In conclusion, it was found polymorphism in the samples MRSA from the analysis of polymorphism of the coagulase gene.