Optimising the efficacy of clofazimine against biofilm-encased Mycobacterium tuberculosis

Abstract

Background: The chemotherapy of tuberculosis (TB) patients is administered for a six to nine- month period consisting of an intensive phase during the first two months with four primary anti- TB drugs, rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and pyrazinamide (PZA),

followed by a continuation phase during the remaining four to seven months with RMP and INH. During the intensive phase the active-replicating organisms (AR) are effectively and rapidly eliminated (99% killing), while the slow-replicating (SR) / non-replicating (NR) populations are targeted during the continuation phase. These latter bacterial populations respond poorly to treatment and are often associated with disease reactivation and relapse in treated patients, highlighting the necessity of identifying effective antimicrobial agents against these bacteria. Clofazimine (CFZ) has demonstrated high antimycobacterial activities against the AR, SR and NR microbial populations in vitro. However, its effects in combination with the primary drugs against these bacteria have not been demonstrated.

Aim and objectives: To investigate the antimycobacterial activity of CFZ in combination with

primary anti-TB drugs against the AR and SR organisms isolated in planktonic and biofilm- forming cultures respectively, by evaluating their inhibitory and bactericidal activities.

Methods: The inhibitory activities of CFZ and three primary anti-TB drugs viz. RMP, INH and EMB were evaluated individually and in combination using minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) determinations by the microtitre Alamar blue assay (MABA) and biofilm formation/ and crystal violet quantification for planktonic and biofilm cultures respectively. The bactericidal activities of these various combinations of the test agents were evaluated using minimum bactericidal concentration (MBC) and fractional bactericidal concentration index (FBCI) determinations by colony-counting procedures. Results: In planktonic cultures, CFZ demonstrated a high inhibitory (MIC: 0.15 μg/mL), but low bactericidal activity (MBC: 5 μg/mL). In combination with primary anti-TB drugs, CFZ demonstrated synergistic inhibitory activities in combination with RMP and INH individually, as well as when the two antibiotics were used together. With respect to bactericidal activity, CFZ exhibited synergistic activity only in a two-drug combination with RMP. Synergistic activities were also demonstrated in a two-drug combination of RIF and INH and in a three-drug combination of these two antibiotics with EMB. However, in biofilm-forming cultures, CFZ demonstrated high inhibitory and bactericidal activities, achieving equal MIC and MBC values of 0.15 μg/mL. All CFZ-containing anti-TB drug combinations exhibited synergistic effects, with high activities being shown in combinations containing RIF and INH. Conclusion: CFZ exhibited synergistic effects in combination with primary anti-TB drugs against both planktonic and biofilm-forming cultures, showing potential benefit in promoting treatment outcome when used in TB chemotherapy.