Journal name: Infection and Drug Resistance Article Designation: Original Research Year: 2018 Volume: 11 Infection and Drug Resistance Dovepress Running head verso: Rizk et al Running head recto: Effects of fluoroquinolones on Babesia parasites open access to scientific and medical research DOI: http://dx.doi.org/10.2147/IDR.S159519 Open Access Full Text Article ORIGINAL RESEARCH Inhibitory effects of fluoroquinolone antibiotics on Babesia divergens and Babesia microti, blood parasites of veterinary and zoonotic importance Mohamed Abdo Rizk1,2 Aim: This study aimed to evaluate the inhibitory effects of fluoroquinolone antibiotics, includ- Mahmoud AbouLaila3 ing enrofloxacin, enoxacin, trovafloxacin, norfloxacin, and ofloxacin, on the in vitro and in vivo Shimaa Abd El-Salam growth of Babesia divergens and Babesia microti parasites, respectively. El-Sayed1,4 Materials and methods: The in vitro and in vivo inhibitory effects of fluoroquinolone Azirwan Guswanto1,5 antibiotics against B. divergens and B. microti, respectively were evaluated using fluorescence- based assay. Additionally, combination therapies of highly effective fluoroquinolone antibiotics Naoaki Yokoyama1 (enrofloxacin, enoxacin, and trovafloxacin) with diminazene aceturate, luteolin, or pyronaridine Ikuo Igarashi1 tetraphosphate were tested on the in vitro cultures of B. divergens. 1 National Research Center for Results: Enrofloxacin, trovafloxacin, and enoxacin were the most effective fluoroquinolones Protozoan Diseases, Obihiro University of Agriculture and against the in vitro growth of B. divergens, followed by norfloxacin and ofloxacin. Further- Veterinary Medicine, Inada-Cho, more, a combination of enoxacin or trovafloxacin with either diminazene aceturate, luteolin, Obihiro, Hokkaido, Japan; or pyronaridine tetraphosphate significantly enhanced the inhibitory effect on the growth ofB. 2Department of Internal Medicine and Infectious Diseases, Faculty of divergens in in vitro cultures. In mice infected by B. microti, enoxacin and diminazene aceturate Veterinary Medicine, Mansoura combination therapy exhibited a potential antibabesial effect. University, Mansoura, Egypt; These results suggest that safe and cheap fluoroquinolone, such as enoxacin, 3Department of Parasitology and Conclusion: Pathology, Faculty of Veterinary might be used for the treatment of clinical cases caused by Babesia spp. in animals or humans. Medicine, Damanhour University, Keywords: Babesia, fluoroquinolones, in vitro, in vivo Damanhour, ElBehera, Egypt; 4Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Mansoura Introduction University, Mansoura, Egypt; Babesia is a protozoan parasite transmitted by ticks; it is a major pathogen that infects 5Veterinary Services Section, Disease the erythrocytes of a wide range of economically valuable animals as well as human Investigation Center, Subang, West 1 Java, Indonesia erythrocytes. Babesiosis is a severe, rapidly fatal disease typified by fever, hemolytic anemia, and hemoglobinuria that causes severe economic losses in the livestock indus- try. 1,2 Babesia microti and Babesia divergens are blood parasites that infect rodents and cattle, respectively, and have zoonotic importance.1 B. microti is the main etiologic agent recognized for babesiosis in humans in the USA and B. divergens is a serious cause of babesiosis in Europe.3 Recently, antibabesial drugs commonly used in veterinary medicine have shown problems regarding parasite resistance and toxicity to the host.4 At the same time, many Correspondence: Ikuo Igarashi of the most effective drugs against B. divergens have been withdrawn from the market National Research Center for Protozoan Diseases, Obihiro University of due to safety or residue problems.3 Moreover, therapeutic failures have been reported in Agriculture and Veterinary Medicine, some severe cases of human babesiosis after the administration of clindamycin, azithro- Inada-Cho, Obihiro, Hokkaido 080-8555, Japan mycin, quinine, and tetracycline.3 Therefore, there is an urgent need to develop more Tel +81 155 49 5641 effective drugs against Babesia parasites that have both veterinary and zoonotic impor- Fax +81 155 49 5643 Email [email protected] tance. Fluoroquinolones are bactericidal drugs that have the ability to interfere with the submit your manuscript | www.dovepress.com Infection and Drug Resistance 2018:11 1605–1615 1605 Dovepress © 2018 Rizk et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://dx.doi.org/10.2147/IDR.S159519 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Rizk et al Dovepress replication of an organism’s DNA by inhibiting pathogen DNA (PYR) (all drugs from Sigma-Aldrich Co.) was prepared as a gyrases and topoisomerases.5 Following this pattern, two gene- 100 mM stock solution and stored at -20°C until use. encoding gyrases (A and B) are present within apicomplexan parasites, with the remarkable exception of Cryptosporidium Evaluation of fluoroquinolone antibiotics sp., and are driven to their essential plastid-like organelles, against B. divergens in vitro the apicoplasts.6 In fact, apicomplexan apicoplast organelles The in vitro Babesia fluorescence assay (BFA) was performed play a critical role in cell survival in the later stages of parasite as previously described by Rizk et al.9 Briefly, Roswell Park evolution.6 Taken together, there is no ortholog to the apicoplast Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich DNA gyrase of apicomplexan parasites in mammals;6,7 thus, Co.) alone or mixed with the indicated concentrations, 0.25, this enzyme provides an attractive target for drug develop- 0.5, 1, 5, and 10 µM for DA or 0.5, 10, 50, 100, and 400 µM ment. Additionally, DNA topoisomerases (Top) are important for other fluoroquinolone antibiotics (enrofloxacin, enoxacin, enzymes required to produce a more relaxed DNA and thus trovafloxacin, norfloxacin, and ofloxacin), was used for the play a critical role in DNA packaging.8 Therefore, in the pres- cultivation of parasitized RBCs (pRBCs) in double 96-well ent study, fluoroquinolone antibiotics, including enrofloxacin, plates (Nunc, Roskilde, Denmark) at 5% hematocrit (HCT) for enoxacin, trovafloxacin, norfloxacin, and ofloxacin, were 4 days in triplicate wells for each concentration of the drug. On selected to evaluate their inhibitory effects against the growth the fourth day of culture, a lysis buffer containing 2×SGI was of B. divergens in vitro and B. microti in vivo. added to each well of the first 96-well plate with gentle mixing. For a viability test, on the fourth day of treatment, 3.5 µL of Materials and methods the culture of untreated pRBCs, treated pRBCs, and uninfected Ethical approval RBCs on the second plate was removed and replaced with All experimental protocols in this study were approved by parasite-free bovine RBCs (3.5 µL) and suspended in 100 µL of the Animal Care and Use Committee, Obihiro University of a fresh growth medium without drugs. Then the plate was incu- Agriculture and Veterinary Medicine (approval no. 27-65). bated at 37°C for the next 4 days. Next, the fluorescence values All experiments were conducted in accordance with the were determined using a fluorescence plate reader (Fluoroskan Fundamental Guidelines for Proper Conduct of Animal Ascent; Thermo Electron Informatics, Philadelphia, PA, USA) Experiment and Related Activities in Academic Research at 485 nm and 518 nm excitation and emission wavelengths, Institutions under the jurisdiction of the Ministry of Educa- respectively. Each experiment was repeated three times. tion, Culture, Sports, Science and Technology, Japan. In vitro drug combination test Parasites Combination therapies of highly effective fluoroquinolone A German bovine strain of B. divergens9 was used for the in antibiotics (enrofloxacin, enoxacin, and trovafloxacin) with vitro experiment and cultivated in purified bovine red blood DA, luteolin, or PYR were tested on the in vitro cultures of cells (RBCs) using a microaerophilic, stationary-phase cul- B. divergens. Fluoroquinolone antibiotics/DA, luteolin, or ture system.9 The Munich strain of B. microti10 was used for PYR combinations (M1, M2, M3, and M4, respectively) were the in vivo experiment. prepared as previously described11,12 with some modifica- tions. Combinations were based on the calculated IC50 values Chemical reagents obtained from the in vitro BFA (Table S1). Three separate SYBR Green I (SGI) nucleic acid stain (10,000×; Lonza, trials were performed, consisting of triplicate experiments Rockland, ME, USA) was stored at –20°C and thawed before for each drug combination over a period of 4 days using 5% use. A lysis buffer consisting of Tris (130 mM; pH 7.5), EDTA HCT. On the fourth day of culture, the fluorescence values (10 mM), saponin (0.016%; W/V), and TritonX-100 (1.6%; were calculated after adding a lysis buffer containing 2×SGI V/V) was prepared in advance and stored at 4°C. Diminazene to each well on the 96-well plate. aceturate (DA; Novartis