Dual β-Lactam Combinations Highly Active against Mycobacterium abscessus Complex In Vitro

Abstract

As a consequence of a growing population of immunocompromised individuals, including transplant recipients and cystic fibrosis patients, there has been a dramatic increase in chronic infections caused by Mycobacterium abscessus complex (MABC) strains that are usually recalcitrant to effective antibiotic therapy. The recent rise of macrolide resistance in MABC has further complicated this clinical dilemma, dramatizing the need for novel agents. The repurposing of current antibiotics is one rapid path from discovery to patient care. In this study, we have discovered that dual β-lactams, and specifically the combination of ceftazidime with either ceftaroline or imipenem, are synergistic and have clinically relevant activities, with MIC₅₀s of 0.25 (ceftaroline with 100 µg/ml ceftazidime) and 0.5 µg/ml (imipenem with 100 µg/ml ceftazidime) against clinical MABC isolates. Similar synergy was observed in time-kill studies against the M. abscessus ATCC 19977 strain using clinically achievable concentrations of either imipenem (4 µg/ml) or ceftaroline (2 µg/ml), as the addition of ceftazidime at concentrations of ≥50 µg/ml showed a persistent bactericidal effect over 5 days. Treatment of THP-1 human macrophages infected with three different M. abscessus clinical isolates supported the in vitro findings, as the combination of 100 µg/ml ceftazidime and 0.125 µg/ml ceftaroline or 100 µg/ml ceftazidime and 0.25 µg/ml imipenem dramatically reduced the CFU counts to near baseline levels of infection. This study's finding that there is synergy between certain β-lactam combinations against M. abscessus infection provides optimism toward identifying an optimum dual β-lactam treatment regimen.IMPORTANCE The emergence of chronic MABC infections among immunocompromised populations and their inherent and acquired resistance to effective antibiotic therapy have created clinical challenges in advancing patients for transplant surgery and treating those with disease. There is an urgent need for new treatment regimens, and the repurposing of existing antibiotics provides a rapid strategy to advance a laboratory finding to patient care. Our recent discoveries that dual β-lactams, specifically the combination of ceftazidime with ceftaroline or ceftazidime with imipenem, have significant in vitro MIC values and kill curve activities and are effective against infected THP-1 human macrophages provide optimism for a dual β-lactam treatment strategy against MABC infections. The unexpected synergistic activities reported in this study create a new path of discovery to repurpose the large family of β-lactam drugs.

Keywords: Mycobacterium abscessus; beta-lactamases; beta-lactams; multidrug resistance.

FIG 1

Phylogenetic analysis and susceptibility testing of M. abscesses complex isolates. The MICs of ≤2, 4, and ≥8 μg/ml were defined as susceptible, intermediate, and resistant to clarithromycin. Acquired clarithromycin resistance was determined at day 3, while inducible clarithromycin resistance was determined at day 14.

FIG 2

In vitro time-kill studies with ceftaroline (A) or imipenem (B), either alone or in combination with different concentrations of ceftazidime. CAZ, ceftazidime; CFT, ceftaroline; IMI, imipenem.

FIG 3

Intracellular activity of ceftaroline (A) or imipenem (B), either alone or in combination with avibactam or avibactam-ceftazidime. Ceftaroline (CFT; 0.125 μg/ml), imipenem (IMI; 0.25 μg/ml), ceftazidime (CAZ; 100 μg/ml), or avibactam (AVI; 4 μg/ml) alone or in different combinations were used. *, compared with no drug group, P < 0.05.

FIG 4

Genetic environment of the bla_Mab gene. The promoter sequence of bla_Mab is embedded in the upstream gene (locus tag MAB_2874). The putative −35 and −10 promoter sequences are underlined in the lower sequence. TSS, transcription start site. The stop codon (TGA) of MAB_2874 is underlined in the upper sequence, while the start codon (ATG) of bla_Mab is shown in bold.

FIG 5

In vitro (A) and intracellular (B) bla_Mab expression during exposure to different combinations of ceftaroline or imipenem, either alone or in combination with ceftazidime and/or avibactam. Ceftaroline (0.125 μg/ml), imipenem (0.25 μg/ml), ceftazidime (100 μg/ml), or avibactam (4 μg/ml) alone or in different combinations was used.