Protective Signatures of Roselle ( Hibiscus sabdariffa L.) Calyx Fractions against Staphylococcus aureus in Drosophila Infection Model Firzan Nainu 1* , M. Natsir Djide 1 , Subehan 1 , Sartini 1 , Tri Puspita Roska 1 , Emil Salim 2, 3 , Takayuki Kuraishi 3 1 Faculty of Pharmacy, Universitas Hasanuddin, Makassar, Indonesia 2 Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Indonesia 3 Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Ishikawa, Japan 1. Introduction The escalation of worldwide infection cases caused by antibiotic-resistant bacteria presents a serious challenge to the health of global population (Levy and Marshall 2004; Gelband et al. 2015). An example of such pathogenic bacteria is Staphylococcus aureus (WHO 2017), especially the strain so called Methicillin-resistant S. aureus (MRSA) (Hassoun et al. 2017). Current guideline in the treatment of S. aureus infection primarily relies on the application of first-line β-lactam antibiotics, such as selected penicillin and cephalosporins derivatives, and in the MRSA-related infections, vancomycin remains the first-choice arsenal to be used in the treatment (Tong et al. 2015). Unfortunately, the increasing number of S. aureus strains that are resistant to available antibiotics has been widely reported (Tong et al. 2015; Hassoun et al. 2017; WHO 2017), critically emphasizes the need to discover new antibacterial agents that are effective to treat the pathogenic mutant strains of S. aureus. At present, directed exploration of natural resources is the main pathway to discover new entities of antibacterial agents with a possible novel mode of action (Lewis 2013; Wright 2014). Of many potential sources to find antibacterial compounds with such characteristics, roselle (Hibiscus sabdariffa L.) calyx is perhaps one of great choices. Roselle calyx extract has been reported to exert antistaphylococcal activity both in vitro (Liu et al. 2005; Alaga et al. 2014) and in vivo (Ahsan et al. 2019). With such promising results in hand, it seems sensible to advance research into the next level: step-by-step assessment on the nature of antibacterial compound(s) contained in the roselle calyx extract. In effort to discover new antibacterial agent(s) from natural resources, the use of inexpensive and practical in vivo model organism become an important subject to consider. In accordance with that, we recently demonstrated the use of fruit fly or vinegar fly (Drosophila melanogaster) infection model to assess the ABSTRACT The rise of antibiotic-resistant Staphylococcus aureus-related clinical cases is an alarming chronicle for global communities. This research was conducted to examine the antistaphylococcal efect of roselle (Hibiscus sabdarifa L.) calyx fractions in the Drosophila model. In the infection experiment, wild-type and immunodefcient Drosophila were pricked with S. aureus and subsequently subjected to fy survivorship and colony-forming assays, in the presence or absence of roselle calyx fractions. The Involvement of immune stimulation in the host antibacterial protection was assessed in vitro using cell-based luciferase reporter assay and in vivo using RT-qPCR analysis on adult fies. A declining rate of fy survivorship and augmentation of bacterial growth were observable in S. aureus- infected wild-type fies but subject to improvement in the presence of roselle calyx fractions. Cell-based analysis revealed the absence of host immune stimulation via Drosophila Toll pathway and roselle calyx fractions-treated immune-defcient fies lacking for components in the Toll pathway were protected from infection-induced early death phenotype and harbored reduced number of S. aureus colonies. Overall, our data confrmed the in vivo anti-staphylococcal activity of roselle calyx fractions in Drosophila infection model and such protective signature was devoid of host immune stimulation. ARTICLE INFO Article history: Received March 14, 2020 Received in revised form September 15, 2020 Accepted September 22, 2020 KEYWORDS: Drug-resistant bacteria, Antistaphylococcal, Hibiscus sabdariffa, roselle fractions, Fruit fly model * Corresponding Author E-mail Address: frzannainu@unhas.ac.id Copyright ©2020 Institut Pertanian Bogor Vol. 27 No. 4, October 2020 306-313 DOI:10.4308/hjb.27.4.306 ISSN: 1978-3019 EISSN: 2086-4094 H A Y AT I Journal of Biosciences