LETTER
The Mycobacterium abscessus complex (MabsC) is a group of multidrug-resistant nontuberculous mycobacteria (NTM) which may cause pulmonary and extrapulmonary infections (1). It consists of three subspecies: Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii. Treatment outcomes are often poor, with clarithromycin susceptibility (largely mediated by the erm41 gene) being one of the key factors influencing treatment outcome (2, 3). Clarithromycin is usually recommended as the key drug in treatment regimens and is used in combination with other antibiotics based on antimicrobial susceptibility testing (4). However, the organism is often resistant to various antibiotics (5), and there is interest in exploring new antimicrobials with potential antimycobacterial activity against MabsC.
In vitro activity of eravacycline has been demonstrated against MabsC (5, 6). Eravacycline is a fluorocycline which is an analogue of tetracyclines. Despite intrinsic resistance to tetracyclines with little inhibition in the usual tested ranges (MIC50 of >16 mg/liter) (5), eravacycline MICs occur between 0.015 and 2 mg/liter. Another antimicrobial of interest is rifabutin. In addition to its having lower MICs against MabsC than other rifamycins, synergy has been demonstrated with clarithromycin, mediated by inhibition of expression of the whiB7 regulator of erm(41) (7, 8). In this study, we explored synergy of eravacycline in combination with rifabutin and clarithromycin.
Checkerboard testing was performed between eravacycline against clarithromycin, rifabutin, clofazimine, and bedaquiline, using cation-adjusted Mueller-Hinton broth (BD Diagnostics, Franklin Lakes, NJ, USA). Antibiotic powders were purchased from Medchem Express (Monmouth Junction, NJ, USA). Testing was performed in 96-well microtiter plates using a final volume of 100 μl, with a final bacterial inoculum size of 5 × 104 CFU. Plates were incubated under ambient conditions at 30°C. The eravacycline-rifabutin combination was incubated to day 5, while the eravacycline-clarithromycin combination was incubated to day 14, as recommended by CLSI for testing inducible clarithromycin resistance. The plates were read visually, with MICs read at 100% inhibition (no visible bacterial growth). The fractional inhibitory concentration index (FICI) was calculated; a FICI of ≤0.5 was defined as synergistic, a FICI of 0.5 of 4 as indifferent, and a FICI of >4 as antagonistic (9).
Twenty-three clinical isolates (10 Mycobacterium abscessus, 10 Mycobacterium massiliense, and three Mycobacterium bolletii isolates) were chosen from a larger collection of isolates for which whole-genome sequencing was performed on the Illumina Hiseq sequencing platform (Illumina, Inc., San Diego, CA, USA). The average sequencing depth was 150×. Species identification was determined by phylogenetic analyses, and multilocus sequence typing (MLST) was performed using the scheme for Mycobacterium abscessus (https://github.com/phac-nml/mab_mabscessus). Isolates with different sequence types were selected to represent genomically diverse isolates.
The results are summarized in Table 1. Eravacycline was neither synergistic nor antagonistic in combination with rifabutin and clarithromycin. Only one isolate had an FICI of ≤0.5 for the eravacycline-rifabutin combination. Despite synergy not being demonstrated on the checkerboard assay, there is still potential for inclusion of eravacycline in treatment regimens for MabsC infections. No antagonism was seen with the key antimicrobial clarithromycin or rifabutin as a potential antibiotic repurposed to treat MabsC. Additional data are required to further explore this with animal models or clinical data.
TABLE 1.
MIC and FICI results for eravacycline tested in combination with rifabutin and clarithromcyina
| Species | Isolate | ST | erm(41) sequevar | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Alone |
In combination (ERV + RFB) |
Alone |
In combination (ERV + CLR) |
||||||||||
| ERV | RFB | ERV | RFB | ERV | CLR | ERV | CLR | ||||||
| M. abscessus | RGM2 | 28 | WT | 0.06 | 1 | 0.03 | 1 | 1.5 | 0.06 | 32 | 0.03 | 32 | 1.5 |
| RGM32 | 62 | T28C | 0.06 | 2 | 0.06 | 1 | 1.5 | 0.03 | 0.5 | 0.06 | 0.5 | 3 | |
| RGM34 | 101 | WT | 0.12 | 1 | 0.12 | 1 | 2 | 0.06 | 32 | 0.03 | 32 | 1.5 | |
| RGM44 | 147 | T28C | 0.12 | 2 | 0.12 | 1 | 1.5 | 0.12 | 0.25 | 0.12 | 0.25 | 2 | |
| RGM60 | 31 | WT | 0.12 | 4 | 0.06 | 1 | 0.75 | 0.12 | 16 | 0.12 | 16 | 2 | |
| RGM66 | 34 | WT | 0.06 | 2 | 0.12 | 1 | 2.5 | 0.06 | 32 | 0.12 | 32 | 3 | |
| RGM72 | 5 | WT | 0.12 | 2 | 0.12 | 1 | 1.5 | 0.12 | 32 | 0.12 | 32 | 2 | |
| RGM121 | 40 | T28C | 0.12 | 2 | 0.03 | 1 | 0.75 | 0.06 | 0.25 | 0.03 | 0.25 | 1.5 | |
| RGM129 | 245 | T28C | 0.12 | 4 | 0.06 | 1 | 0.75 | 0.12 | 0.25 | 0.03 | 0.25 | 1.25 | |
| RGM218 | 23 | T28C | 0.06 | 4 | 0.06 | 1 | 1.25 | 0.12 | 0.5 | 0.12 | 0.5 | 2 | |
| M. bolletii | RGM7 | 6 | WT | 0.12 | 4 | 0.12 | 1 | 1.25 | 0.12 | 0.25 | 0.03 | 0.25 | 1.25 |
| RGM217 | 54 | WT | 0.25 | 4 | 0.25 | 1 | 1.25 | 0.03 | 0.25 | 0.03 | 0.25 | 2 | |
| RGM238 | 10 | WT | 0.12 | 2 | 0.06 | 1 | 1 | 0.12 | 0.25 | 0.03 | 0.25 | 1.25 | |
| M. massiliense | RGM19 | 76 | `NA | 0.25 | 4 | 0.06 | 1 | 0.5 | 0.06 | 0.25 | 0.03 | 0.25 | 1.5 |
| RGM51 | 29 | `NA | 0.12 | 2 | 0.03 | 1 | 0.75 | 0.12 | 0.25 | 0.03 | 0.25 | 1.25 | |
| RGM68 | 186 | `NA | 0.06 | 4 | 0.06 | 1 | 1.25 | 0.12 | 0.25 | 0.03 | 0.25 | 1.25 | |
| RGM112 | 12 | `NA | 0.12 | 2 | 0.06 | 1 | 1 | 0.12 | 0.25 | 0.03 | 0.25 | 1.25 | |
| RGM122 | 62 | `NA | 0.25 | 4 | 0.12 | 1 | 0.75 | 0.12 | 0.25 | 0.03 | 0.25 | 1.25 | |
| RGM123 | 32 | `NA | 0.06 | 4 | 0.06 | 1 | 1.25 | 0.06 | 0.25 | 0.03 | 0.25 | 1.5 | |
| RGM139 | 4 | `NA | 0.5 | 8 | 0.5 | 1 | 1.125 | 0.25 | 0.25 | 0.06 | 0.25 | 1.25 | |
| RGM163 | 7 | `NA | 0.06 | 8 | 0.06 | 1 | 1.125 | 0.12 | 0.25 | 0.06 | 0.25 | 1.5 | |
| RGM167 | 5 | `NA | 0.12 | 4 | 0.06 | 1 | 0.75 | 0.06 | 0.25 | 0.06 | 0.25 | 2 | |
| RGM233 | 100 | `NA | 0.12 | 2 | 0.03 | 1 | 0.75 | 0.03 | 0.25 | 0.03 | 0.25 | 2 | |
ST, Sequence type; ERV, eravacycline; RFB, rifabutin; CLR, clarithromycin, FICI, fractional-inhibitory-concentration index; WT, wild type; NA, not applicable due to truncated erm41.
ACKNOWLEDGMENTS
We have no conflicts of interest to declare.
This work was supported by the NUS Yong Loo Lin School of Medicine Pitch For Funds Grant and by the National Medical Research Council (NMRC, Singapore) via the Collaborative Solutions Targeting Antimicrobial Resistance Threats in Health System Antimicrobial Resistance Research Grant (CoSTAR-HS/ARGSeedGrant/2019/03).
Contributor Information
Ka Lip Chew, Email: ka_lip_chew@nuhs.edu.sg.
Ayush Kumar, University of Manitoba.
REFERENCES
- 1.Lee M-R, Sheng W-H, Hung C-C, Yu C-J, Lee L-N, Hsueh P-R. 2015. Mycobacterium abscessus complex infections in humans. Emerg Infect Dis 21:1638–1646. doi: 10.3201/2109.141634. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Kwak N, Dalcolmo MP, Daley CL, Eather G, Gayoso R, Hasegawa N, Jhun BW, Koh W, Namkoong H, Park J, Thomson R, Van Ingen J, Zweijpfenning SMH, Yim J. 2019. Mycobacterium abscessus pulmonary disease: individual patient data meta-analysis. Eur Respir J 54:1801991. doi: 10.1183/13993003.01991-2018. [DOI] [PubMed] [Google Scholar]
- 3.Chew KL, Cheng JWS, Osman NH, Lin RTP, Teo JWP. 2017. Predominance of clarithromycin-susceptible Mycobacterium massiliense subspecies: characterization of the Mycobacterium abscessus complex at a tertiary acute care hospital. J Med Microbiol 66:1443–1447. doi: 10.1099/jmm.0.000576. [DOI] [PubMed] [Google Scholar]
- 4.Haworth CS, Banks J, Capstick T, Fisher AJ, Gorsuch T, Laurenson IF, Leitch A, Loebinger MR, Milburn HJ, Nightingale M, Ormerod P, Shingadia D, Smith D, Whitehead N, Wilson R, Floto RA. 2017. British Thoracic Society guidelines for the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD). Thorax 72:ii1–ii64. doi: 10.1136/thoraxjnl-2017-210927. [DOI] [PubMed] [Google Scholar]
- 5.Chew KL, Octavia S, Go J, Ng S, Tang YE, Soh P, Yong J, Jureen R, Lin RTP, Yeoh SF, Teo J. 2021. In vitro susceptibility of Mycobacterium abscessus complex and feasibility of standardizing treatment regimens. J Antimicrob Chemother 76:973–978. doi: 10.1093/jac/dkaa520. [DOI] [PubMed] [Google Scholar]
- 6.Kaushik A, Ammerman NC, Martins O, Parrish NM, Nuermberger EL. 2019. In vitro activity of new tetracycline analogs omadacycline and eravacycline against drug-resistant clinical isolates of Mycobacterium abscessus. Antimicrob Agents Chemother 63:e00470-19. doi: 10.1128/AAC.00470-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Aziz DB, Go ML, Dick T. 2020. Rifabutin suppresses inducible clarithromycin resistance in Mycobacterium abscessus by blocking induction of whiB7 and erm41. Antibiotics 9:72. doi: 10.3390/antibiotics9020072. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Cheng A, Tsai Y-T, Chang S-Y, Sun H-Y, Wu U-I, Sheng W-H, Chen Y-C, Chang S-C. 2019. In vitro synergism of rifabutin with clarithromycin, imipenem, and tigecycline against the Mycobacterium abscessus complex. Antimicrob Agents Chemother 63:e02234-18. doi: 10.1128/AAC.02234-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Odds FC. 2003. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 52:1. doi: 10.1093/jac/dkg301. [DOI] [PubMed] [Google Scholar]