Research Article Microbiology International Journal of Pharma and Bio Sciences ISSN 0975-6299 SCREENING OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) FROM SPUTUM SAMPLES PRIYANKA SHARMA * Dr. K. N Modi institute of pharmaceutical education and research, Modi nagar, Ghaziabad, Uttar pradesh, India. ABSTRACT Staphylococcal infections are a group of infections caused by the bacterium Staphylococcus but most infections are caused by a type called Staphylococcus aureus. Bacteria are constantly evolving because their genes are constantly changing. The result of this is that some of the bacteria develop more resistance to a certain antibiotic than others. Methicillin-resistant Staphylococcus aureus is a strain of Staphylococcus aureus who developed resistance to methicillin and is responsible for several infections in humans which cannot be treated easily. It is resistant to many antibiotics and is called as multidrug-resistant Staphylococcus aureus and oxacillin-resistant Staphylococcus aureus. MRSA have resistance because of the widespread use of antibiotics, genetic selection and our dislike of tablets. Most MRSA infections are skin infections but it is also responsible for life-threatening bloodstream infections, pneumonia and surgical site infections. In the present study, a total number of 21 respiratory/sputum samples were collected and screened for methicillin resistant Staphylococcus aureus by biochemical and antibiotic sensitivity tests. Out of 21 samples, 10 were observed as methicillin resistant Staphylococcus aureus. It was found that MRSA shows highly variable antibiotic resistance. All MRSA shows 100% resistance to penicillin but were recorded sensitive to vancomycin. Thus penicillin and methicillin were not found to be effective against MRSA. The study suggests that vancomycin may be used as the drug of choice for treating multidrug resistant MRSA infections. KEYWORDS: Sputum, Staphylococcus aureus, MRSA, MSSA, Antibiotic sensitivity. PRIYANKA SHARMA Dr. K. N Modi institute of pharmaceutical education and research, Modi nagar, Ghaziabad, Uttar pradesh, India. *Corresponding author B - 496
INTRODUCTION Staphylococci are gram-positive cocci that occur in grape like clusters. Their ability to develop resistance to penicillin and other antibiotics enhances their importance as a human pathogen, especially in the hospital environment (1). Staphylococcus aureus means golden cluster seed or the seed gold and also known as golden staph and Orostaphira is a facultative anaerobic, Grampositive coccus and is the most common cause of Staphylococcus infections. It is frequently a part of the skin flora found in the nose and on skin. About 20% of the human population is carrier of S. aureus (2). Some strains of Staphylococcus aureus are capable of producing staphyloxanthin (a golden colored carotenoid pigment). This pigment acts as a virulence factor, primarily by being a bacterial antioxidant which helps the microbe evade the reactive oxygen species which the host immune system uses to kill pathogens (3, 4). Staphyloxanthin may be key to the ability of S. aureus to survive immune system attacks. Drugs designed to inhibit the bacterium's production of staphyloxanthin may weaken it and renew its susceptibility to antibiotics. In fact, because of similarities in the pathways for biosynthesis of staphyloxanthin and human cholesterol, a drug developed in the context of cholesterol-lowering therapy used to block S. aureus pigmentation and disease progression in a mouse infection model (3, 5). S. aureus can cause a range of illnesses from minor skin infections, such as pimples, impetigo, boils or furuncles, cellulitis, folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomylitis, endocarditis, toxic shock syndrome (TSS), chest pain, bacteremia, and sepsis. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections, often causing postsurgical wound infections (6). S. aureus appears as grape-like clusters when viewed through a microscope, and has large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates (7). S. aureus is catalase-positive. It can produce the enzyme catalase and able to convert hydrogen peroxide (H 2 O 2 ) to water and oxygen. Thus, catalase test is useful to distinguish staphylococci from enterococci and streptococci (8). The coagulase test is used to differentiate Staphylococcus aureus from coagulase negative staphylococci. S. aureus produces two forms of coagulase (i.e. bound coagulase and free coagulase). Bound coagulase, otherwise known as "clumping factor", can be detected by carrying out a slide coagulase test, and free coagulase can be detected using a tube coagulase test (9). Methicillin-resistant S. aureus (MRSA) includes those strains that have acquired a gene giving them resistance to methicillin and essentially all other beta-lactam antibiotics. MRSA was first reported in 1961, soon after methicillin was introduced into human medicine to treat penicillin-resistant staphylococci. This group of organisms has since emerged as a serious concern in human (10, 11, 12 13, medicine 14). Resistance to methicillin is mediated via the mec operon, part of the staphylococcal cassette chromosome mec (SCCmec). Resistance is conferred by the meca gene, which codes for an altered penicillin binding protein that has a lower affinity for binding β-lactams such as penicillins, cephalosporins and carbapenems. This allows for resistance to all β-lactam antibiotics and obviates their clinical use during MRSA infections. As such, the glycopeptides vancomycin is often used against MRSA (15). Spread of S. aureus including MRSA is through human-to-human contact, although recently some veterinarians have discovered that the infection can be spread through pets (16). MATERIALS AND METHODS Total no. of 21 sputum samples were collected for the screening of Methicillin resistant Staphylococcus aureus in sterile manner. After collection streaking of samples was done on to the blood agar media plates for the growth. Gram staining, Biochemical tests and Antibiotic sensitivity tests were performed to B - 497
confirm the presence of Methicillin resistant Staphylococcus aureus. Staining Staining was done by Gram staining method to confirm the presence of Gram positive and Gram negative bacteria. Morphology of bacteria was also observed. Biochemical test Biochemical tests (catalase and coagulase) were performed to determine the activity of a particular enzyme produced by microorganism in a sample which helps in its identification. Catalase test was performed by using 3% hydrogen peroxide to observe the effervescences after mixing with a loopfull of culture for catalase positive. Coagulase tests were performed to observe the clumping action for coagulase positive. Coagulase tests were conducted in both slide and tube by mixing of diluted plasma with a loopfull of culture. Antibiotic Sensitivity test for screening of MRSA Antibiotic sensitivity tests were carried out by disc diffusion, E test and with methicillin agar. These tests were used to determine the susceptibility of bacteria to antibiotics and to know which antibiotic will be most successful in treating a bacterial infection. Screening of MRSA using Disk diffusion Test Muller-Hinton Agar (MHA) media was used for the screening of MRSA. It is a solid medium used for susceptibility testing of pathogens. Bauer et al., (1966) have recommended this medium as routine methods for disk susceptibility testing (17). Muller-Hinton Agar plates were swabbed with broth of Staphylococcus aureus. Then disks of methicillin antibiotic were placed on the agar surface. After 24 hours of incubation at 37 C, the clear zones were observed. Screening of MRSA using E Test E test was also used for the antimicrobial susceptibility to screen the MRSA. The system comprises a predefined antibiotic gradient which is used to determine the minimum inhibitory concentration (MIC). Muller-Hinton plates (with 2% NaCl) were swabbed with broth of Staphylococcus aureus. Methicillin strips were applied by using sterile forcep with the MIC scale facing upward. After 24 hours of incubation at 37 C zones of inhibition were observed. Screening of MRSA using Methicillin Agar Methicillin Agar was used for the screening of MRSA. It was prepared by adding 6µg/ ml of methicillin and 4% NaCl in Mueller Hinton Agar to make them selective. RESULTS AND DISCUSSION For the screening of Methicillin resistant Staphylococcus aureus, 21 sputum samples were collected in a sterile manner and processed by the inoculation of sample on to the blood agar plate by streaking. After the incubation at 37 C for 48 hours growth was observed. Colonies were smooth, glistening and surrounded by a narrow zone of haemolysis on Blood agar. Pigmentation is a characteristic of this species when grown aerobily and ranges from cream through buff to Gold. The Gram staining showed that the bacteria observed in colonies were Gram positive cocci. When catalase and coagulase tests were performed, they were found to be catalase +ve by showing effervescences and Coagulase +ve by showing clumping action (Fig 1, 2). Antibiotic Sensitivity test for screening of MRSA was done by using Disk diffusion Test, E Test (Fig 3, 4) and with Methicillin Agar (Table 1). Out of 21 Staphylococcus aureus, 10 (47.6%) Methicillin Resistant Staphylococcus aureus (MRSA) were observed (Graph 1). The remaining strains were Methicillin Sensitive Staphylococcus aureus (MSSA). MRSA show highly variable antibiotic resistance. All MRSA show 100% resistance to penicillin, 73.07% cefazolin, 57.6% ciprofloxacin, 7.69% chloramphenicol and 0% to vancomycin (Graph 2). Complete resistance was recorded against penicillin. However, all MRSA strains recorded were sensitive to vancomycin. B - 498
Figure 1 Showing Catalase +ve with effervescences. Figure 2 Showing Coagulase +ve with clumping action. Figure 3 Showing Disk diffusion test. B - 499
Figure 4 Strip 1 shows positive and strip 2 shows negative E Test. Table 1 MRSA screening with methicillin, disk diffusion and E test methods. Samples MRSA Screening with methicillin Disk diffusion test E Test S-1 NG S S S-2 NG S S S-3 G R R S-4 G R R S-5 G R R S-6 G R R S-7 NG S S S-8 G R R S-9 NG S S S-10 NG S S S-11 NG S S S-12 NG S S S-13 NG S S S-14 G R R S-15 G R R S-16 NG S S S-17 G R R S-18 NG S S S-19 G R R S-20 G R R S-21 G R R S =Sensitive, R =Resistant, G = Growth, NG=No growth Graph 1 Showing MRSA percentage in respiratory samples B - 500
Graph 2 Antimicrobial resistance analysis of MRSA isolates. CONCLUSION The persons who have strong immune systems have the ability to fight with Staphylococcus aureus infections and show only mild symptoms. However, the persons who do not have such a strong immune or who have undergone surgery can develop more serious problems. In more susceptible persons, Staphylococcus aureus shows many other effects like boils, impetigo, abscesses, septic wounds, heart valve problems, toxic shock syndrome and may also results death in more severe cases. In the present study, a total number of 21 Staphylococcus aureus were isolated from Respiratory/Sputum samples. Out of 21 Staphylococcus aureus, 10 (47.6%) Methicillin Resistant Staphylococcus aureus (MRSA) were observed. The Epidemiology of MRSA is gradually changing since its emergence was reported. Moreover, the association of Multidrug resistance with MRSA has added to the problem. Beta-lactam antibiotics like Penicillin, Methicillin were not found to be effective against MRSA. Vancomycin seems to be the only antimicrobial agent which showed 100% sensitivity and may be used as the drug of choice for treating multidrug resistant MRSA infections. REFERENCES 1. R. Ananthanarayan, and C.K.J Paniker. Staphylococcus. In: Textbook of Microbiology, 7 th Edn, Orient Longman, 2005, 7, 192. 2. Ogston A, On Abscesses: Classics in Infectious Diseases. Rev Infect Dis, 6 (1): 122 28, (1984). 3. Liu GY, Essex A, Buchanan JT, Datta V, Hoffman HM., Bastian JF, Fierer J and Nizet V, Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. J Exp Med, 202 (2): 209 15, (2005). 4. Clauditz A, Resch A, Wieland KP, Peschel A and Gotz F, Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infection and immunity, 74 (8): 4950 3, (2006). 5. Liu CI, Liu GY, Song Y, Yin F, Hensler ME, Jeng WY, Nizet V, Wang AH and Oldfield E, A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence. Science, 319 (5868): 391 94, (2008). 6. Bowersox and John, Experimental Staph Vaccine Broadly Protective in Animal Studies. NIH. Archived from the original B - 501
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