Pak. J. Pharm. Sci., Vol.31, No.5(Suppl), September 2018, pp.2143-2148 2143 Assessment of killing kinetics assay and bactericidal mechanism of crude methanolic bark extract of Casuarina equisetifolia Yousra Shafiq 1 , Syed Baqir Shyum Naqvi 2 , Ghazala H Rizwani 3,4 , Tanveer Abbas 5 , Huma Sharif 6 , Huma Ali 1 , Muhammad Arif Asghar 1 , Rabia Bushra* 7 , Farya Zafar 8 , Saima Abdein 1 and Ambreen Huma 9 1 Department of Pharmaceutics, Faculty of Pharmacy, Jinnah Sindh Medical University, Karachi, Pakistan 2 Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University Karachi, Pakistan 3 Hamdard University, Karachi, Pakistan 4 Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, Pakistan 5 Department of Microbiology, University of Karachi, Karachi, Pakistan 6 Department of Pharmacognosy, Faculty of Pharmacy, Jinnah Sindh Medical University, Karachi, Pakistan 7 Department of Pharmaceutics, Faculty of Pharmacy, Dow University of Health Sciences, Karachi, Pakistan 8 Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, Pakistan 9 Department of Pharmacognosy, Faculty of Pharmacy, Ziauddin University, Karachi, Pakistan Abstract: Casuarina equisetifolia L. is an important medicinal plant widely used to treat various diseases particularly ulcers, diabetes, cough, diarrhea and many infectious and skin diseases. The aim of this research study was to examine the killing mechanism and killing kinetics assay of methanolic bark extract of C. equisetifolia against some highly resistant human pathogens. The comparison on antibacterial activity of extract was firstly done with six different well reputed antibiotics using disk diffusion method. The broth dilution method was used to measure the MIC and MBC values. The mechanism of killing was identified by scanning electron microscopy (SEM) technique. Results showed that higher inhibitory zones were produced by methanolic plant extract than that of some tested antibiotics. The lower MIC and MBC values indicated the antibacterial potency of plant extract. The extract of C. equisetifolia produced a more drop in optical density of S. aureus, MRSA B. subtilis and S. epidermidis up to 12 hrs. The complete destruction of the cell membrane of MRSA was observed after 12 h treatment with plant extract. It is concluded that crude bark extract of C. equisetifolia is potent antimicrobial agent and produced both bacteriostatic and bactericidal effects. Its killing time was extremely faster especially against MRSA. The cell membrane rapturing is a suggested killing mechanism of plant extract. Keywords: Antimicrobial activity, Casuarina equisetifolia, killing mechanism, killing kinetic assay, Methicillin resistant Staphylococcus aureus. INTRODUCTION Antimicrobial resistance is renowned as one of the greatest burden to human health worldwide (Walker et al., 2009). One of the widespread factors linked with the antibiotic resistance is consumption behavior of the global population. Improper or injudicious exercise of these agents in a variety of disciplines like medicine, dentistry, veterinary medicine, food industry and agriculture may be considered to play a task in coming out of multi-resistant bacterial strains (Epstein et al., 2000). Therefore global alarm of antibiotic resistance is a big dare for current medicine, and it seems to return public health to the “Pre- antibiotic era” where no successful or appropriate treatment against bacterial infections was accessible (Cars et al., 2008). High expenditure of manufacturing of synthetic compounds, increasing resistance towards presently used antimicrobials along with many side effects, call for an action to reduce this problem. Plants are well known for antimicrobial activities. In the battle of microbial infections, plants are considered as strong tool due to presence of anti-infective agents such as emetine, quinine, berberine, tannins, terpenoids, alkaloids and flavonoids (Cowan 1999). Casuarina equisetifolia L. (Casuarinaceae) is a beautiful tree, with needle shaped branch lets and with dropping branches. Different parts of this plant are used such as bark reported as anti- acne as well as to treat diarrhea, dysentery, lotion for swelling headache and fever, antimicrobial and anticancer (Essien et al., 2016, Gumgumjee and Hajar 2012, Shafiq et al., 2014). The knowledge regarding its bacterial killing kinetics and killing mechanism against different pathogenic strains is insufficient. In order to promote C. equisetifoliaas a bacteriostatic and bactericidal plant, pathogenic strains of different microorganisms were kept for observation to determine its antimicrobial activity. Therefore, this study was planned to investigate the antibacterial actions of bark extract of C. equisetifolia against some highly *Corresponding author: e-mail: rabia_pharmacist@hotmail.com