Detection of mecA Gene and Identification of Potential Methicillin-resistant Staphylococcus Aureus in Hospital Wastewater Samples

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important causes of hospital infections worldwide. Methicillin-resistant S. aureus (MRSA) tends to be resistant to multiple antibiotics. High-level resistance to antibiotics is caused by the mecA gene, which encodes an alternative penicillin-binding protein, PBP 2a. The present study was aimed to detect mecA gene in potential methicillin-resistant Staphylococcus aureus (MRSA) isolates in clinical wastewater. Three hospital wastewater samples were collected, and the bacteria were isolated in mannitol salt agar (MSA) medium and Baird-parker agar medium. Gram staining and biochemical tests were performed. PCR amplification of extracted DNA from these bacterial isolates was done with 16S rRNA universal primers, specific primers of S. aureus and mecA gene primers to screen the clinical bacterial isolates. Sequencing of mecA gene amplicon was also done. The sequences were analyzed using BLAST (NCBI) and EMBOSS Needle tool (EMBL-EBI). Moreover, antibiotic resistance was tested at the levels of 50, 100, 200 and 300 μg/mL ampicillin. PCR amplification with all the primers screened was resulted expected bands for the isolates from Polgahawela and Chilaw wastewater. Two bacterial isolates of Polgahawela hospital effluent were able to grow at 200 μg/mL ampicillin. However, sequence analysis of amplified mecA gene product of these two bacterial isolates showed sequence similarity with the penicillin-binding protein (mecA) gene of Staphylococcus aureus strain and methicillin-resistance gene region of Staphylococcus sciuri 28C with 95% and 96% identity, respectively. Pairwise alignment results proved 89.6% sequence similarity between the two sequences. In conclusion, potential methicillin-resistant staphylococcus aureus (MRSA) along with Staphylococcus sciuri was able to detect only in the clinical effluent collected from Polgahawela base hospital.