Anti-Mycobacterium avium complex activity of clarithromycin, rifampin, rifabutin, and ethambutol in combination with adenosine 5′-triphosphate

Highlights

• In vitro anti-MAC activity of CLA+ rifamycin and CLA+ EMB was increased by ATP.

• ATP-mediated synergistic activity was expressed in a strain-dependent manner.

• In vitro regrowth of drug-treated bacteria was delayed by combined use of ATP.

Abstract

We previously reported that adenosine 5′-triphosphate (ATP) inhibited the growth of various bacteria, including mycobacteria, Staphylococcus, and Pseudomonas, without damaging bacterial surface structures. Notably, ATP's antibacterial activity was found to be attributable to its iron-chelating ability. ATP exhibited combined effects with some antimicrobials against Mycobacterium intracellulare and methicillin-resistant S. aureus, suggesting its usefulness as an adjunctive drug in the chemotherapy against certain intractable infections. In this study, we examined detailed profiles of the anti-Mycobacterium avium complex (MAC) activity of some antimicrobial agents, including clarithromycin (CLA), rifampin (RIF), rifabutin (RBT), and ethambutol (EMB), in combination with ATP. It was found that the anti-MAC activity of CLA + RIF, CLA + RBT, and CLA + EMB was markedly potentiated in a strain-dependent manner. In this case, the onset of the regrowth of antimicrobial agent-treated mycobacteria during cultivation was significantly delayed in the presence of ATP, indicating the usefulness of ATP as an adjunctive drug in chemotherapy against MAC infections.

Introduction

Clinical management of Mycobacterium avium complex (MAC) infection is difficult, since it is frequently encountered in immunocompromised hosts, particularly AIDS patients, and MAC organisms are moderately to highly resistant to common anti-tuberculosis drugs such as isoniazid, ethambutol (EMB), pyrazinamide, and rifampin (RIF) (Inderlied et al., 1993). Although some drugs including clarithromycin (CLA), azithromycin, and rifabutin (RBT) are fairly effective in controlling MAC disseminated infection (Benson, 1997, Inderlied et al., 1993), treatment of pulmonary MAC infections is still difficult even with the use of multi-drug regimens containing these drugs (Benson, 1997, Inderlied et al., 1993, Tomioka, 2000). Although some new antimicrobial agents active against MAC, such as diarylquinoline, ketolides, pyrimidines, and pyrroles, are being developed (Tomioka et al., 2008), limited numbers of new drugs have been subjected to clinical studies for chemotherapy against MAC infections. Thus, at present, it appears that devising potent anti-MAC administration protocols based on the combination of ordinary antimycobacterial drugs with adjunctive agents is more practical than awaiting the development of new antimycobacterial drugs.

It is known that adenosine 5′-triphosphate (ATP) up-regulates macrophage antimycobacterial activity in a purinergic receptor X₇ (P2X₇)-dependent manner (Idzko et al., 2014, Lammas et al., 1997). The ATP/P2X₇-mediated activation of the antimicrobial functions of macrophages is associated with the up-regulation of phospholipase D, leading to the potentiation of phagosomal maturation, induction of apoptosis, and stimulation of intracellular translocation and concomitant activation of cytosolic phospholipase A₂ (Fairbairn et al., 2001, Kusner and Adams, 2000, Placido et al., 2006, Tomioka et al., 2005). In this context, we recently found that ATP inhibited the growth of various bacteria, such as Staphylococcus, Pseudomonas, and some mycobacteria including MAC and M. tuberculosis, without damaging bacterial surface structures, while Escherichia coli and Klebsiella pneumoniae were resistant to ATP's antimicrobial activity (Tatano et al., 2015). Using gene technology, we newly established an enterobactin-deficient (entB⁻) mutant from ATP-resistant K. pneumoniae, and observed the recovery of ATP susceptibility in the entB⁻ mutant. Since, in the case of K. pneumoniae, enterobactin plays a major role among K. pneumoniae's siderophores (enterobactin, aerobactin, and yersiniabactin) in the bacterial acquisition of Fe³⁺ ions (El Fertas-Aissani et al., 2013), this finding strongly indicates that ATP's antibacterial activity is attributable to its iron-chelating ability (Tatano et al., 2015). Notably, ATP exhibited significant combined effects with some antimicrobials against methicillin-resistant S. aureus and M. intracellulare, suggesting its usefulness as an adjunctive drug in chemotherapy against certain intractable infections (Tatano et al., 2015). These findings encouraged us to examine the detailed profiles of the potentiation of the antimicrobial activity of various anti-MAC drugs (AMDs) in combination with ATP. It was found that the anti-MAC activity of the three drug combinations consisting of two drugs, including CLA + RIF (CLA/RIF), CLA + RBT (CLA/RBT), and CLA + EMB (CLA/EMB), was markedly potentiated dependent on test MAC strains.