ANITA DWI ANGGRAINI, Magister Biology, Faculty of Biology, University of Jenderal Soedirman. Polymorphism analysis of the coA (coagulase) gene in isolates of methicillin-resistant S. aureus with AluI restriction sites. Supervisors : Dr. Daniel Joko Wahyono, M.Biomed and Dr. Hendro Pramono, M.Si
MRSA is a condition in which diseases caused by S. aureus are resistant to beta-lactic group of antibiotics such as penicillin. MRSA occurs due to genetic changes caused by antibiotic exposures. It is predicted that infections mostly occur to patients who have previously experienced colonization of S. aureus or MRSA. 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 (Saha et al., 2008).
The prevalence of MRSA in various hospitals in the world ranges from 2-70% with an average of 20% (Oliveira et al., 2002). 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% (Mehraj et al., 2014). In overall Asia, MRSA prevalence has reached 70% (Wahid, 2007), while MRSA publication and prevalence in Indonesia are still very limited and difficult to obtain. Noviana reports that in 2003 MRSA prevalence at Atmajaya Hospital of Jakarta has reached 47%. Yuwono repots that in 2010 MRSA at Dr. Moh. Hoesin Provincial Public Hospital of Palembang in 2010 has reached 46%. S. aureus is able to produce coagulase which is a protein enzyme that can crumple oxalate or citrate plasma. The bacteria that form the coagulase are considered to have the potential to become invasive pathogens. Coagulase makes S. aureus able to avoid phagocytosis and inhibit the penetration of antibiotics with the formation of a protective wall layers in the form of fibrin. (Tiwari dkk, 2005).
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.
The analytical studies on methicillin-resistant S. aureus coA gene polymorphism using AluI restriction enzyme have been conducted on methichillin-resistant S. aureus isolate population in Iran (Mahmoudi et al., 2016), India (Babu et al., 2014; Himabindu et al., 2009 ), and Europe (Hookey et al., 1997), which result in different RFLP patterns. In Indonesia, studies on methicillin-resistant S. aureus coA gene polymorphism with AluI restriction enzyme have never been conducted before that the researcher is interested in conducting polymorphism analysis of the coA (coagulase) gene in isolates of methicillin-resistant S. aureus with AluI restriction sites.
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 result shows that methicillin resistant S. aureus coA gene in patients at Dr. Soetomo hospital of Surabaya is polymorphic as the most dominant allele proportion is only 64%. Hartl (1988) states that a gene is considered polymorphic when the most dominant allele proportion is less than 95% and PCR-RFLP of CoA gene shows that all 25 samples 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%, it is then respectively followed by RFLP IV by 24% and RFLP II by 8%. Meanwhile, RFLP III pattern has the lowest proportion by only 4%. Those four patterns show that CoA gene from methicillin-resistant S. aureus bacteria collected from the isolate samples have polymorphisms, because all of the pattern RFLP genes the coA have genetic variation sequence. The research is first study which proves that S. aureus resistant methichillin in clinical samples on human beings in Indonesia dominated pattern RFLP which was not digested by AluI restriction enzyme.
The study conducted by Hookey et al. (1997) suggest that the coA gene polymorphism on methichillin-resistant S. aureus population in Europe shows that RFLP I pattern which is undigested by AluI restriction enzyme may become RFLP pattern with the lowest proportion of only about 2%. A similar study is also conducted by Mahmoudi et al. (2016) stating similar finding that coA gene polymorphism in methichillin-resistant S. aureus population in Iran suggests that RFLP I pattern which is undigested by AluI restriction enzyme may become RFLP pattern with the lowest proportion of only about 2%. In India, Himabindu et al. (2009) also state that the results of RFLP coA gene patterns are divided into 31. Those thirty-one pattern variations show that all samples may be well digested by AluI. A similar latest study conducted by Babu et al. (2014) state that in India, the study of S. aureus coA gene polymorphism on the population in India shows that RFLP S. aureus patterns are divided into 5. Those five pattern variations also show that all samples may be well digested by AluI.
There are several variation differences of RFLP MRSA patterns in coA gene found in this study when compared with those found by the other researchers as shown that the strains of methichillin-resistant S. aureus is polymorphic. The polymorphic shown by the sequential changes of AluI enzyme introduction in coA gene since there is a point mutation. These sequencial changes may cause genetic diversity of S. aureus coA genes. The presence of genetic diversity in coA gene is in accordance with the results of research conducted Gosh et al. (1992) and Mahmoudi et al. (2016) stating that the coA gene polymorphic is also due to the differences in sequence 3 ' of coA gene regional variable on S. aureus strains which may result in various amino acid sequential encoding in coA gene.
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 coA gene is extremely helpful in treating various infections caused by methicillin-resistant S. aureus based on its genotype.
Keywords : Staphylococcus aureus, coA gene, PCR-RFLP, AluI