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Detection of Methicillin-resistant Staphylococcus Aureus (mrsa) from Animal and Human Origin in Bangladesh by Polymerase Chain Reaction

M. A. Islam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. M. Alam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. S. Uddin
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

N. Kobayashi
Department of Hygiene, School of Medicine, Sapporo Medical University, Sapporo 060, Japan

M. U. Ahmed
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

Methicillin-resistant Staphylococcus aureus (MRSA) is defined by the presence of the mecA gene, which is considered to have been transferred horizontally from unknown bacterial species to S. aureus. The mecA gene which encodes an additional β-lactam-resistant penicillin-binding protein (PBP), termed PBP-2a (PBP-2’) with reduced binding affinity for β-lactam compounds. We investigated distribution of the mecA gene in a total of 94 clinical strains of S. aureus isolated from both man and animal admitted in Bangladeshi medical hospital as well as Veterinary clinic. The mecA gene was detected by PCR in 25% of human clinical isolates of S. aureus, whereas not a single mecA gene was detected in animal isolates of S. aureus.

Keywords: Methicillin-resistant Staphylococcus aureus (MRSA), Polymerase Chain Reaction (PCR), Animal & Human

Location:

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity:

Objectives:

Therefore, this research will focus on the distribution and prevalence of MRSA in animal and human origin in Bangladesh.

Methodology:

The bacteria was isolated & identified by cultural characteristics, colony morphology, Gram’s stain and biochemical tests. MRSA was detected by PCR, as described previously by Kobayashi et al. (1994). A total of 100 μl TNE buffer was taken in eppendorf tube in which bacterial sample was added and mixed by vortexing, centrifuged at 10000 rpm for 1 minute. The supernatant was removed by micropipette & added 10 μl achromopeptidase enzyme (disrupting solution) with the pellet, mixed well by pipetting and incubated the tube at 40-420 C in water bath for 10 minutes. Removed from water bath, added 50 μl of 0.5 M KOH in the tube and mixed by vortex, kept at room temperature for 5 minutes. Finally 50 μl of 1M-Tris-Hcl (PH 6.76) was added and mixed by vortex, centrifuged at 10000 rpm for 1 minute and collected supernatant which contains DNA used for PCR A total amount of 79 μl de-ionized distilled water (DDW) was taken to the eppendorf tube, added 10 μl X10 reaction/PCR buffer (Roche Diagnostics, Germany) & 8 μl of 2.5 mM dNTP (Takara, Japan) to the tube. Then added 2 μl mecA primer,1 μl supernatant DNA sample and 0.5μl Taq Polymerase (5 units/μl, Roche Diagnostics, Germany) to the tube. Gently mixed by vortex and then kept tubes in thermal cycler. A thermal cycler was used to amplification of DNA. The cycling program included 30 cycles of a denaturing step at 940C for one minute, an annealing step at 550C for one minutes, and an extension step at 720C for two minutes. After completion of cycling program reactions were held at 40C. Based on the nucleotide sequences of mecA gene (Song et al., 1987 and Berger-Bächi et al., 1989), the oligonucleotide primers were designed (Table 1) and target genes were synthesized by PCR. After electrophoresis, the gel was taken out carefully from the gel chamber and the gel gently placed on the UV transilluminator in the dark chamber of the image documentation system. The UV light of the system was switched on; the image was viewed on the monitor, focused and saved in a dictate, as well as printed on thermal paper. The positive sample was detected by visualized band on the gel.

Source of Information: Bangl. J. Vet. Med. (2011). 9 (2): 161 – 166

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Achievement/Findings:

The prevalence of MRSA differs strongly throughout the countries. In the present study, no MRSA in animals was found while in human the isolation rate is 25%. In 1999-2000, 20% of the European blood isolates were MRSA which is agreement with present study. Another study from Bangladesh in human reported an isolation rate of MRSA as 12.5%, which also indicates that the incidence of MRSA in our country is increasing day by day. The increasing incidence of MRSA observed in this study might be due to the fact that our specimens were taken from tertiary hospital where there is no authentic antibiotic policy to treat infectious patients. As a result, indiscriminate use of antibiotics is not less common. In addition, hospital environment are not adequately hygienic. Overcrowding of patients and attendants favors the spread of infectious agents. So hospitals acquired infection either in surgery or in medical wards are quite high. Due to infection, patients stay becomes prolong in hospital keeping under antibiotic therapy. All these factors mentioned above might be sufficient cause to increase the acquisition of resistance property among strains of S. aureus.

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