Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Tue, 18 Dec 2018 17:39:40 Combination therapy with thioridazine and dicloxacillin combats meticillin-resistant Staphylococcus aureus infection in Caenorhabditis elegans Marianne Ø. Poulsen, 1,2 Lone Schøler, 3 Anette Nielsen, 2 Marianne N. Skov, 1 Hans Jørn Kolmos, 1 Birgitte H. Kallipolitis, 2 Anders Olsen 3 and Janne K. Klitgaard 1,2 Correspondence Janne K. Klitgaard jkklitgaard@health.sdu.dk Received 6 January 2014 Accepted 9 June 2014 1 Institute of Clinical Research, Research Unit of Clinical Microbiology, University of Southern Denmark, Odense, Denmark 2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark 3 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark The shortage of drugs active against meticillin-resistant Staphylococcus aureus (MRSA) is a growing clinical problem. In vitro studies indicate that the phenothiazine thioridazine (TZ) might enhance the activity of the b-lactam antibiotic dicloxacillin (DCX) to a level where MRSA is killed, but experiments in simple animal models have not been performed. In the present study, we introduced Caenorhabditis elegans infected by S. aureus as an in vivo model to test the effect of TZ as a helper drug in combination with DCX. Because TZ is an anthelmintic, initial experiments were carried out to define the thresholds of toxicity, determined by larval development, and induction of stress-response markers. No measurable effects were seen at concentrations of less than 64 mg TZ l ”1 . Seven different MRSA strains were tested for pathogenicity against C. elegans, and the most virulent strain (ATCC 33591) was selected for further analyses. In a final experiment, full-grown C. elegans were exposed to the test strain for 3 days and subsequently treated with 8 mg DCX l ”1 and 8 mg TZ l ”1 for 2 days. This resulted in a 14-fold reduction in the intestinal MRSA load as compared with untreated controls. Each drug alone resulted in a two- to threefold reduction in MRSA load. In conclusion, C. elegans can be used as a simple model to test synergy between DCX and TZ against MRSA. The previously demonstrated in vitro synergy can be reproduced in vivo. INTRODUCTION The treatment of meticillin-resistant Staphylococcus aureus (MRSA) is becoming increasingly problematic worldwide. Many strains are multi-drug resistant and there is a shortage of active drugs because of slow progress in developing new antibiotics (Fischbach & Walsh, 2009). Therefore, there is an urgent need for new strategies with which to manage MRSA. MRSA is resistant to dicloxacillin (DCX) and other penicillinase-resistant penicillins, which are the drugs of choice for the treatment of meticillin-susceptible S. aureus (MSSA). We have previously shown that the neuroleptic drug thioridazine (TZ) acts in synergy with DCX to an extent that restores the susceptibility of MRSA to DCX and increases the susceptibility of MSSA to DCX in vitro (Klitgaard et al., 2008; Poulsen et al., 2013). Furthermore, we have analysed the molecular mechanisms underlying the synergy between TZ and DCX (Bonde et al., 2011; Klitgaard et al., 2008; Thorsing et al., 2013). Most impor- tantly, TZ was shown to affect the cell wall biosynthesis pathway by interfering with the formation of the penta- glycine bridge of peptidoglycan precursors, resulting in enhanced sensitivity to DCX (Thorsing et al., 2013). From a clinical point of view, the combination of TZ and DCX looks promising. Before this combination can be tested in a clinical setting, however, animal experiments are needed. The nematode Caenorhabditis elegans has been established as a host model for S. aureus pathogenesis, in which nematodes fed S. aureus die over the course of days as a Abbreviations: CA, community associated; DCX, dicloxacillin; ER, endoplasmic reticulum; HA, healthcare associated; HSP, heat-shock protein; MRSA, meticillin-resistant Staphylococcus aureus; MSSA, meticillin-susceptible Staphylococcus aureus; NGM, nematode growth medium; TSB, Trypticase soy broth; TZ, thioridazine. Journal of Medical Microbiology (2014), 63, 1174–1180 DOI 10.1099/jmm.0.071837-0 1174 071837 G 2014 The Authors Printed in Great Britain