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Meca Gene and Methicillin-Resistant Staphylococcus Aureus (MRSA) Isolated from Dairy Farms in East Java, Indonesia

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Meca Gene and Methicillin-Resistant Staphylococcus Aureus (MRSA) Isolated from Dairy Farms in East Java, Indonesia BIODIVERSITAS ISSN: 1412-033X Volume 21, Number 8, August 2020 E-ISSN: 2085-4722 Pages: 3562-3568 DOI: 10.13057/biodiv/d210819 MecA gene and methicillin-resistant Staphylococcus aureus (MRSA) isolated from dairy farms in East Java, Indonesia SANCAKA CHASYER RAMANDINIANTO1, ASWIN RAFIF KHAIRULLAH2, MUSTOFA HELMI EFFENDI3,4, 1Program of Animal Diseases and Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga. Jl. Mulyorejo, Surabaya 60115, East Java, Indonesia 2Doctoral Program in Veterinary Science, Faculty of Veterinary Medicine, Universitas Airlangga. Jl. Mulyorejo, Surabaya 60115, East Java, Indonesia 3Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga. Jl. Mulyorejo, Surabaya 60115, East Java, Indonesia 4Halal Research Center, Universitas Airlangga. Jl. Mulyorejo, Surabaya 60115, East Java, Indonesia. Tel./fax.: +62-31-5915551, email: [email protected] Manuscript received: 27 May 2020. Revision accepted: 13 July 2020. Abstract. Ramandinianto SC, Khairullah AR, Effendi MH. 2020. MecA gene and methicillin-resistant Staphylococcus aureus (MRSA) isolated from dairy farms in East Java, Indonesia. Biodiversitas 21: 3562-3568. Milk Borne Disease (MBD) can be caused by a variety of pathogenic bacteria, one of which is Staphylococcus aureus which has a large impact on aspects of public health. The therapy used to treat staphylococcal infection is Oxacillin preparations that can inhibit bacterial wall synthesis, but the adaptation of the mecA gene to staphylococcal cassette chromosome mec (SCCmec) causes the emergence of strains of methicillin-resistant S. aureus (MRSA). The purpose of this study was to detect the level of MRSA strain contamination in dairy cows in East Java by comparing the mecA gene, Oxacillin, and Cefoxitin Disc Diffusion Methods and Oxacillin Resistance Screen Agar (ORSA) detection methods. A total of 150 cow's milk samples were taken at 3 village dairy farms in East Java, samples were added to the enrichment media Buffer Pepton Water (BPW) and then isolates were planted and purified using Mannitol Salt Agar (MSA). The detection of MRSA was carried out by the Kirby Bauer disc diffusion preparation Cefoxitin 30 μg and Oxacillin 30 μg then confirmed by ORSA and the presence of mecA gene by the polymerase chain reaction (PCR) method. The results showed that from a total of 92 S. aureus isolates using Oxacillin disc test, 24 resistant isolates were obtained, using Cefoxitin disc test, 17 isolates were obtained, and using the ORSA test 18 MRSA isolates were obtained. MRSA isolates tested by PCR obtained evidence of 2 isolates of mecA gene. It can be concluded that the Oxacillin disc test was the highest sensitivity for detecting MRSA strain isolate, however, mecA gene was the golden standard to detect MRSA on the dairy farms. Keywords: Cefoxitin disc, milk-borne disease, mecA gene, MRSA, Oxacillin disc, ORSA INTRODUCTION aureus (MRSA) has occurred in humans but has now been detected in animals (Kumar and Prasad 2010; Chajęcka- Milk borne diseases (MBD) are a very important Wierzchowska et al. 2015). MRSA resistance to beta- problem in the public health sector which can be caused by lactam antibiotics is caused by various mechanisms, one of a variety of pathogenic bacteria, one of which is which is the production of unusual penicillin-binding Staphylococcus aureus. S. aureus opportunistic pathogens protein (PBP), which forms PBP2 thereby weakening the that are often found in humans and animals, these bacteria affinity for the antibiotic β-lactam expressed by the mecA can cause a diverse spectrum of diseases from minor skin gene (Katayama et al. 2000). MecA gene detection using infections to systemic such as pneumonia and meningitis the polymerase chain reaction (PCR) method is the gold (Jangra and Singh 2010). Some researchers suggest that S. standard for detecting MRSA, but cannot be done in all aureus can be transmitted to humans through clinical laboratories due to various facilities, capabilities contamination of milk, unprocessed milk, and milk and costs (Fernandes et al. 2005). The difficulty of using products (Seifu et al. 2004; Swetha et al. 2017). S. aureus PCR in an effort to detect the presence of MRSA can be is commonly found in the skin and mucosa of ruminants, reduced by using Cefoxitin disc diffusion, Oxacillin disc which have sub-clinical or clinical mastitis which is a diffusion combination of Oxacillin Resistance Screen Agar source of contamination in dairy products (Sasidharan et al. (ORSA) (Tyansningsih et al. 2019; Decline et al. 2020). 2011). The purpose of this study was to detect and evaluate the A research report reveals that the presence of multiple level of MRSA contamination in dairy cow milk in East antibiotic resistance from S. aureus creates new problems Java and to compare the phenotypic detection method using for the world of health practitioners and researchers screening using Cefoxitine disc diffusion, Oxacillin disc (Motamedi et al. 2010). There are studies on phenotypic diffusion combination of Oxacillin Resistance Screen Agar and genotypic antibiotic resistance stating that since 1962 (ORSA) and confirm genotypically using PCR to detect the methicillin-resistant Staphylococci (MRS) have been found MecA gene. The sensitivity and specificity of the test will where the first case of methicillin-resistant Staphylococcus show the effectiveness and ease of application of the RAMANDINIANTO et al. – MecA gene and methicillin-resistant Staphylococcus aureus (MRSA) 3563 MRSA strain detection method. Also, this research Swab S (AKD 10903610549). The swab was streaked information is very important to support strategic and evenly on the surface of the Muller Hinton agar medium technical decision making by relevant institutions for (Oxoid, CM0337). Cefoxitin 30 μg and Oxacillin 30 μg mitigation and prevention of impacts on aspects of public were placed side by side with a distance of 4.5 cm on health in food safety. Muller Hinton agar medium which had been inoculated with isolate and then incubated 37 oC for 24 hours and measured the inhibition zone. In the anefoxitin disc MATERIALS AND METHODS diffusion test inhibition zone ≤21mm is methicillin- resistant (MR) isolate, whereas in Oxacillin disc diffusion Ethical clearance test isolates with an inhibition zone ≤10mm is MR. Raw milk was used in this study, hence ethical clearance was not necessary. Raw milk samples were Oxacillin resistance screen agar test collected from three dairy farms in East Java Province, Oxacillin Resistance Screen Agar test (ORSA Test) is Indonesia, namely Batu, Pasuruan, and Probolinggo carried out referring to the Clinical and Laboratory districts. Standards Institute guidelines aimed at confirming MRSA which is MR from Oxacillin and Cefoxitin Disc Diffusion Sampling Methods (CLSI 2018; Decline et al. 2020). Isolates were Total samples of 150 dairy cows were collected from 3 taken by several colonies to be used as a suspension of 0.5 Village Unit Cooperatives in Probolinggo, Batu, and Mc Farland and then by using Sterile Cotton Swab S Pasuruan areas during October-November 2019. Dairy (Onemed, AKD 10903610549) and making smears on the cows milk collected directly from milk can and as many as Oxacillin Screen Agar Base (HiMedia Pvt. Ltd., M1415) 15 mL were collected in centrifuge tubes of 50 mL added with Oxacillin Resistant Selective Supplement (Biologix, BD-T0034). Samples were taken 1 mL (Supplements, HiMedia Pvt. Ltd., FD191). aseptically to be put into 10 mL containing 4 mL of buffered water peptone buffer media (Oxoid, CM0509) Detection of the mecA gene using Syringe 3CC (AKD 20902900277) then incubated at All confirmed S. aureus isolates were MRSA by the 37oC for 24 hours with an incubator (Isuzu Model 2-2195, ORSA test were tested using PCR to detect the presence of Jica) (Thaker et al. 2013). the mecA gene (Rahmaniar et al. 2020). The DNA extraction process was carried out according to the Bacteria isolation and identification QIAamp DNA Mini Kit protocol (51304 & 51306), where Samples produced from enrichment media were previously the isolate was purified first on Mannitol Salt cultured and purified on Mannitol Salt Agar media Agar (HiMedia Pvt. Ltd, M118) and inoculated on Muller (HiMedia Pvt.Ltd, M118) and incubated at 37 oC for 24 Hinton agar (Oxoid, CM0337). The primers used namely hours. Identification was done by examination based on mecA F: 5 '-GAA ATG GAA CGT CCG ATA A-3' and morphological cultural characteristics, than microscopic mecA R: 5 '-CCA ATT CCA CAT TGT TTC CTA A-3' examination using Gram's method of staining which shows (Rajabiani et al. 2014; Rahmaniar et al. 2020). The master Gram-positive bacteria in the form of coccus and clustering mixture uses GoTaq® Green Master Mix (Promega, (Effendi et al. 2018). Biochemical tests were carried out to 9PIM712) which is a premixed ready-to-use solution confirm the S. aureus species with the catalase test and containing Taq DNA polymerase, dNTPs, MgCl2, and coagulase test (Effendi et al. 2019). Catalase tests were buffers reaction. DNA was amplified using a Thermal carried out by dripping hydrogen peroxide (H2O2) 3% on Cycler T100 machine (Bio-Rad, 186-1096) with an initial clean glass objects and mixing with ose of the 1 colony denaturation step of 4 min at 94 °C than 35 cycles at 94°C (Tyasningsih et al. 2019). Coagulase test was carried out for 1 min, annealing at 62 °C for 1 min, then the extension with Coagulase slide test was given 50 µl rabbit blood at 72°C for 45s. The final extension is carried out for 5 min plasma dripped on a glass object, then mixed with 1 ose of at 72°C. Amplicons were processed with electrophoreses, a bacterial colony, and Coagulase tube test using 200 µl where the gel will be visualized in ultraviolet illumination blood plasma was added with as much as 3-4 isolate (Rahmaniar et al. 2020). Positive tests showed PCR colonies then incubated at 37oC for 24 hours (Effendi et al.
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