Short Communication For reprint orders, please contact: reprints@futuremedicine.com Modulatory effects of verapamil in rifampicin activity against Mycobacterium tuberculosis Katiany R Caleff-Ferracioli* ,1 , Rosilene F Cardoso 1 , Jo˜ ao VP de Souza 1 , Let´ ıcia S Murase 1 , Pedro H Canezin 1 , Regiane BL Scodro 1 , Vera L ´ ucia D Siqueira 1 & Fernando R Pavan** ,2 1 Laboratory of Medical Bacteriology, Department of Clinical Analysis & Biomedicine, State University of Maringa, Parana, Brazil 2 Department of Biological Sciences, School of Pharmaceutical Sciences, Paulista State University, Araraquara, Sao Paulo, Brazil *Author for correspondence: katianyrcf@gmail.com ***Author for correspondence: fernandopavan@fcfar.unesp.br Aim: To evaluate modulatory effect of verapamil (VP) in rifampicin (RIF) activity and its effect in effux pumps (EPs) transcript levels in Mycobacterium tuberculosis. Materials & methods: RIF and VP minimal inhibitory concentration, combinatory effect and detection of mutations were determined in 16 isolates. EPs transcript levels were determined in four isolates by real-time PCR after exposure to drugs. Results: VP showed good combinatory effect among RIF-resistant isolates. This effect was also observed in the relative transcript levels of EPs, mainly after 72 h of exposure, depending on the EP gene, genotype and the resistance profle of the isolate. Conclusion: Additional regulatory mechanisms in the EP activities, as well as, interactions with other drug-specifc resistance mechanisms need further investigation in M. tuberculosis. First draft submitted: 9 October 2018; Accepted for publication: 13 December 2018; Published online: 16 January 2019 Keywords: effux pump inhibitor • gene regulation • multidrug transporters • Mycobacterium tuberculosis • real- time PCR • resistance • rifampicin • synergism • tuberculosis • verapamil The emergence and spread of Mycobacterium tuberculosis-resistant phenotypes are a public health concern nowadays, owing to a smaller number of therapeutic options and high mortality rates in tuberculosis (TB). Worldwide, TB is one of the top ten causes of death from a single infectious agent (above HIV/AIDS). It is estimated that 10 million people developed TB disease and 1.6 million died of TB in 2017 [1]. Although resistance to any anti-TB drug poses a challenge for a successful treatment, rifampicin (RIF) resistance (RIF-R) is the most problematic. RIF is effective against both actively growing and dormant bacilli, and this characteristic greatly shortens the standard anti-TB treatment [2]. Usually, RIF-R TB is treated in the same way as multidrug-resistant TB (MDR-TB), with second-line anti-TB drugs. These drugs are poorly tolerated, often causing further toxic side effects. Furthermore, extensively drug-resistant (XDR) M. tuberculosis strains may emerge during second-line treatment because of poor tolerance and a lack of patient compliance [3]. Resistance to RIF is usually caused by mutations in the rpoB gene, responsible for structural changes on the drug target [4]. Nonetheless, in some (around 5%) clinical isolates, resistance cannot be explained by the presence of known gene mutations, indicating the existence of other mechanisms underlying this phenotype [5,6]. One of such alternative mechanism is the activity of efflux pump (EP) systems. These pumps act by diminishing the intracellular concentration of a given drug, and thus reduce the susceptibility of the bacillus [7]. EPs can not only extrude a broad range of compounds owing to their polysubstrate specificity but also can favor the survival of the microorganism allowing it to continue to multiply and eventually exhibit stochastic mutations that might impart in high level resistance [8]. The upregulation of EP genes in the absence of drug pressure confirms the important role of EPs in the acquisition and maintenance of drug resistance in M. tuberculosis [9]. One way of countering EP-mediated resistance is the use of drugs that can inhibit these systems, namely efflux pump inhibitors (EPIs). To date, EPIs have been used to further elucidate the role of EPs in the development of drug resistance in mycobacteria [10–13] and have been considered as potential adjunctive therapy for TB [14,15]. EPIs, Future Microbiol. (Epub ahead of print) ISSN 1746-0913 10.2217/fmb-2018-0277 C  2019 Future Medicine Ltd