MECHANISMS Holarrhena antidysenterica Extract and Its Steroidal Alkaloid, Conessine, as Resistance-Modifying Agents Against Extensively Drug-Resistant Acinetobacter baumannii Thanyaluck Siriyong, 1 Sasitorn Chusri, 2 Potjanee Srimanote, 3 Varomyalin Tipmanee, 4 and Supayang Piyawan Voravuthikunchai 1 Emergence and spread of antibiotic-resistant Acinetobacter baumannii have become a major public health concern. This study was designed to investigate the efficacy of Holarrhena antidysenterica extract and its major steroidal alkaloid conessine as resistance-modifying agents (RMAs) on the susceptibility of A. baumannii to novobiocin and rifampicin. A significant synergistic activity of both the extract and conessine in combination with either novobiocin or rifampicin with fractional inhibitory concentration index £0.5 was demonstrated. Fluorescent dyes and different efflux pump inhibitors were used to further investigate the synergism. Increase in the uptake of 1-N-phenylnaphthylamine in the bacterial cells treated with the extract and conessine was not observed indicating that both substances did not act as permeabilizers. With regard to efflux pump inhibition, no accumulation in ethidium bromide (EtBr) was noticed suggesting that the AdeABC pump was not involved. In contrast, accumulation in Pyronin Y was significantly increased ( p < 0.05) demonstrating that the synergism was due to interference with the AdeIJK pump. Study on frequencies of the spontaneous mutational resistance to the extract in combination with antibiotics demonstrated attenuation in drug-resistant organisms. Thus, H. antidysenterica extract and conessine as RMAs may offer a combinatory therapy to restore antibiotic susceptibility in the extensively drug-resistant A. baumannii. Introduction A cinetobacter baumannii has emerged as an impor- tant opportunistic pathogen frequently associated with nosocomial outbreaks worldwide. 17 Multidrug-resistant (MDR) A. baumannii has been implicated as the cause of serious infections such as ventilator-associated pneumonia, blood- stream infection, meningitis, urinary tract infection, and wound infection. During the past decade, antibiotic resistance among A. baumannii has substantially increased. Currently, infections with MDR A. baumannii greatly limit therapeutic options leading to an increase in morbidity and mortality. 10,29 Carbapenems remain the antibiotics of choice for the treat- ment of A. baumannii infections. However, carbapenem- resistant A. baumannii is now increasingly reported worldwide. 19 Colistin, an old antibiotic from the polymyxin group, has become the last resort for treatment of MDR A. baumannii. In addition, colistin demonstrates adverse effects regarding nephrotoxicity and neurotoxicity. Recently, colistin-resistant A. baumannii has already been reported. Therefore, com- bination therapy of colistin with other antibiotics might be an applicable alternative. Many in vitro, in vivo, and clinical studies have shown synergistic effects of colistin in com- bination with rifampicin for MDR or extensively drug- resistant (XDR) A. baumannii. 4 In addition, a recent study confirmed the synergistic colistin/rifampicin combination against heteroresistant isolates and prevented the develop- ment of colistin-resistant mutants. 32 However, moderate hepatic cytolysis has been noticed in clinical study. 25 The search for new compounds with biological properties is viewed as an important path toward fighting and reducing antibiotic resistance. 1 Holarrhena antidysenterica (L.) Wall., belonging to the Apocynaceae family, is an important medicinal plant used in Ayurvedic, Unani, and Thai traditional medicines for the treatment of amoebic dysentery and di- arrhea. H. antidysenterica barks are composed of alkaloids, 1 Department of Microbiology and Excellence Research Laboratory on Natural Products, Faculty of Science and Natural Product Research Center of Excellence, Prince of Songkla University, Hat Yai, Thailand. 2 Faculty of Traditional Thai Medicine and Excellence Research Laboratory on Natural Products, Faculty of Science and Natural Product Research Center of Excellence, Prince of Songkla University, Hat Yai, Thailand. 3 Graduate Program, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand. 4 Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat Yai, Thailand. MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2015.0194 1