Matt D Johansen

Wassim Daher

Françoise Roquet-Banères

Clément Raynaud

Matthéo Alcaraz

Florian P Maurer

Laurent Kremer
email: laurent.kremer@irim.cnrs.fr

Rifabutin is bactericidal against intracellular and extracellular forms of Mycobacterium abscessus

Keywords: Mycobacterium abscessus, rifabutin, macrophage, zebrafish, therapeutic activity

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Nontuberculous mycobacteria (NTM) are environmental mycobacteria. Among all NTM, Mycobacterium avium and Mycobacterium abscessus represent the most frequent pathogens associated with pulmonary disease [START_REF] Johansen | Non-tuberculous mycobacteria and the rise of Mycobacterium abscessus[END_REF]. M. abscessus is a rapidly growing NTM of increasing clinical significance, particularly in cystic fibrosis (CF) patients [START_REF] Cowman | Non-tuberculous mycobacterial pulmonary disease[END_REF]. In CF patients, infection with M. abscessus correlates with a more rapid decline in lung function and can represent an obstacle to subsequent lung transplantation [START_REF] Jönsson | Molecular epidemiology of Mycobacterium abscessus, with focus on cystic fibrosis[END_REF][START_REF] Esther | Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis[END_REF][START_REF] Catherinot | Acute respiratory failure involving an R variant of Mycobacterium abscessus[END_REF]. From a taxonomical view, the species currently comprises three subspecies: M. abscessus subsp. abscessus (designated hereafter M. abscessus), M. abscessus subsp. bolletii (designated hereafter M. bolletii) and M. abscessus subsp. massiliense (designated hereafter M. massiliense) [START_REF] Adekambi | Reinstating Mycobacterium massiliense and Mycobacterium bolletii as species of the Mycobacterium abscessus complex[END_REF]. These subspecies exhibit different clinical outcomes and drug susceptible profiles to antibiotic treatments [START_REF] Koh | Clinical significance of differentiation of Mycobacterium massiliense from Mycobacterium abscessus[END_REF].

M. abscessus strains can exhibit either a smooth (S) or rough (R) morphotype as a consequence of the presence or absence, respectively, of bacterial surface glycopeptidolipids (GPL) [START_REF] Johansen | Non-tuberculous mycobacteria and the rise of Mycobacterium abscessus[END_REF][START_REF] Howard | Spontaneous reversion of Mycobacterium abscessus from a smooth to a rough morphotype is associated with reduced expression of glycopeptidolipid and reacquisition of an invasive phenotype[END_REF][START_REF] Gutiérrez | Glycopeptidolipids, a doubleedged sword of the Mycobacterium abscessus complex[END_REF][START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF].

These morphological distinctions are associated with important physiological differences. S variants are more hydrophilic than R variants, enabling increased sliding motility and the capacity to form biofilms [START_REF] Howard | Spontaneous reversion of Mycobacterium abscessus from a smooth to a rough morphotype is associated with reduced expression of glycopeptidolipid and reacquisition of an invasive phenotype[END_REF][START_REF] Gutiérrez | Glycopeptidolipids, a doubleedged sword of the Mycobacterium abscessus complex[END_REF][START_REF] Bernut | Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members[END_REF], while the aggregative R variants possess a high propensity to produce large bacterial cords [START_REF] Bernut | Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members[END_REF][START_REF] Sánchez-Chardi | Demonstration of cord formation by rough Mycobacterium abscessus variants: implications for the clinical microbiology laboratory[END_REF]. While S and R variants can be viewed as two representatives of the same isolate, which can co-exist and evolve differently in response to host immunity, they express different pathophysiological traits [START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF]. S variants are typically less virulent than the R variants [START_REF] Bernut | Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members[END_REF][START_REF] Nessar | Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response[END_REF][START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF], the latter being more frequently associated with severe lung diseases and persisting for years in CF patients [START_REF] Jönsson | Molecular epidemiology of Mycobacterium abscessus, with focus on cystic fibrosis[END_REF][START_REF] Catherinot | Acute respiratory failure involving an R variant of Mycobacterium abscessus[END_REF]. Importantly, an S-to-R transition within the colonized host [START_REF] Catherinot | Acute respiratory failure involving an R variant of Mycobacterium abscessus[END_REF][START_REF] Park | Clonal diversification and changes in lipid traits and colony morphology in Mycobacterium abscessus clinical isolates[END_REF] is linked to genetic polymorphisms within the GPL biosynthetic/transport locus [START_REF] Park | Clonal diversification and changes in lipid traits and colony morphology in Mycobacterium abscessus clinical isolates[END_REF][START_REF] Pawlik | Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus[END_REF]. Moreover, differences in the susceptibility to drug candidates have been identified between S and R variants [START_REF] Madani | Cyclipostins and Cyclophostin analogues as multitarget inhibitors that impair growth of Mycobacterium abscessus[END_REF][START_REF] Lavollay | In vitro activity of cefoxitin and imipenem against Mycobacterium abscessus complex[END_REF], highlighting the need for the improved evaluation of new compounds/drug regimens against both morphotypes. Treatment of M. abscessus lung disease remains particularly challenging, largely due to intrinsic resistance to wide panel of antimicrobial agents, including most antitubercular drugs such as rifampicin (RIF) [START_REF] Nessar | Mycobacterium abscessus: a new antibiotic nightmare[END_REF][START_REF] Van Ingen | Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria[END_REF][START_REF] Brown-Elliott | Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria[END_REF][START_REF] Lopeman | Mycobacterium abscessus: Environmental bacterium turned clinical nightmare[END_REF]. The extensive resistome of M. abscessus results from a low permeability of the cell wall, absence of drug-activating systems, induction of efflux pumps and production of a wide panel of drug-modifying enzymes [START_REF] Nessar | Mycobacterium abscessus: a new antibiotic nightmare[END_REF][START_REF] Lopeman | Mycobacterium abscessus: Environmental bacterium turned clinical nightmare[END_REF][START_REF] Luthra | The role of antibiotic-target-modifying and antibioticmodifying enzymes in Mycobacterium abscessus drug resistance[END_REF]. In addition, mutations in genes encoding drug targets can result in acquired drug resistance further complicating therapy [START_REF] Johansen | Non-tuberculous mycobacteria and the rise of Mycobacterium abscessus[END_REF][START_REF] Wu | NTM drug discovery: status, gaps and the way forward[END_REF]. Treatment of infections caused by M. abscessus require prolonged courses of multiple antibiotics, usually combining a macrolide (azithromycin or clarithomycin), a -lactam (imipenem or cefoxitin) and an aminoglycoside (amikacin) [START_REF] Griffith | An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases[END_REF][START_REF] Floto | US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of nontuberculous mycobacteria in individuals with cystic fibrosis: executive summary[END_REF][START_REF] Daley | Treatment of nontuberculous mycobacterial pulmonary disease: an official ATS/ERS/ESCMID/IDSA clinical practice guideline[END_REF]. Additional drugs, such as tigecycline or clofazimine, are often added to strengthen the regimen, particularly in response to toxic side effects or unsatisfactory clinical response [START_REF] Wallace | Clinical experience in 52 patients with tigecycline-containing regimens for salvage treatment of Mycobacterium abscessus and Mycobacterium chelonae infections[END_REF]. Despite intensive chemotherapy, treatment success rates typically remain around 25-40% in the case of macrolide resistance, which occurs in at least 40-60% of clinical isolates [START_REF] Roux | Comparing Mycobacterium massiliense and Mycobacterium abscessus lung infections in cystic fibrosis patients[END_REF].

Therefore, there is an urgent clinical need for new drug regimens with improved efficacy [START_REF] Daniel-Wayman | Advancing translational science for pulmonary nontuberculous mycobacterial infections. A road map for research[END_REF]. While the current drug pipeline against M. abscessus remains poor, it has recently been fueled with the discovery of several active hits and the development of repurposed drugs [START_REF] Wu | NTM drug discovery: status, gaps and the way forward[END_REF]. Among the latter, screening of libraries of approved pharmaceuticals revealed that rifabutin (RFB), a rifamycin related to the poorly active rifampicin (RIF), shows activity against M. abscessus [START_REF] Aziz | Rifabutin is active against Mycobacterium abscessus complex[END_REF][START_REF] Pryjma | Rifabutin acts in synergy and is bactericidal with frontline Mycobacterium abscessus antibiotics clarithromycin and tigecycline, suggesting a potent treatment combination[END_REF]. RIF, along with many other rifamycins, is inactivated by the ADP-ribosyltransferase (Arr Mab ) encoded by MAB_0591, which ribosylates the drug at the C23 hydroxyl position [START_REF] Rominski | Intrinsic rifamycin resistance of Mycobacterium abscessus is mediated by ADP-ribosyltransferase MAB_0591[END_REF]. RFB has also been reported to be as active as clarithromycin in immune-compromized NOD/SCID mice infected with M. abscessus [START_REF] Dick | Rifabutin is active against Mycobacterium abscessus in mice[END_REF].

However, most studies on RFB have been carried out on either S or R variants (when reported), rendering results sometimes difficult to interpret and/or to compare. Due to the co-existence of S and R variants in patients [START_REF] Park | Clonal diversification and changes in lipid traits and colony morphology in Mycobacterium abscessus clinical isolates[END_REF] and the presence of each variant in different compartments (S residing mostly in macrophages and R growing also in the form of intra-or extracellular cords), it is essential to address the activity of RFB on isogenic S/R pairs in both in vitro and in vivo studies.

The present study aimed to describe and compare the activity of RFB against S and R M. abscessus complex strains in vitro and ex vivo in a macrophage infection model. Due to the importance of cording, considered as a marker of severity of the infection with the R variant, we also investigated the efficacy of RFB in a zebrafish model of infection.

MATERIALS AND METHODS

Mycobacterial strains and growth conditions. M. abscessus CIP104536 T , M. bolletii CIP108541 T and M. massiliense CIP108297 T reference strains and clinical isolates from CF and non-CF patients were reported previously [START_REF] Singh | In vitro evaluation of a new drug combination against clinical isolates belonging to the Mycobacterium abscessus complex[END_REF][START_REF] Halloum | Resistance to thiacetazone derivatives active against Mycobacterium abscessus involves mutations in the MmpL5 transcriptional repressor MAB_4384[END_REF]. Strains were routinely grown and maintained at 30°C in Middlebrook 7H9 broth (BD Difco) supplemented with 0.05% Tween 80 (Sigma-Aldrich) and 10% oleic acid, albumin, dextrose, catalase (OADC enrichment; BD Difco) (7H9 T/OADC ) or on Middlebrook 7H10 agar (BD Difco) containing 10% OADC enrichment (7H10 OADC ) and in the presence of antibiotics, when required. For drug susceptibility testing, bacteria were grown in Cation-Adjusted Mueller-Hinton Broth (CaMHB; Sigma-Aldrich). RFB was purchased from two independent commercial sources (Adooq Bioscience and Selleckchem) and dissolved in DMSO.

Drug susceptibility testing.

The minimal inhibitory concentrations (MIC) were determined according to the CLSI guidelines [START_REF] Woods | Susceptibility testing of mycobacteria, nocardiae and other aerobic actinomycetes: approved standardSecond Edition[END_REF]. The broth micro-dilution method was used in CaMHB with an inoculum of 5x10 6 CFU/mL in exponential growth phase. The bacterial suspension was seeded in 100 µL volumes in all of the wells of a 96-well plate, except for the first column, to which 198 µL of the bacterial suspension was added. In the first column, 2 µL of drug at its highest concentration was added to the first well containing 198 µL of bacterial suspension. Two-fold serial dilutions were then carried out and the plates were incubated for 3-5 days at 30°C. MICs were recorded by visual inspection. Assays were completed in triplicate in three independent experiments.

Growth inhibition kinetics.

To monitor growth inhibition of M. abscessus CIP104536 T S and R, 96-well plates were set-up as for MIC determination and serial dilutions of the bacterial suspensions exposed to increasing concentrations of RFB were plated on LB agar plates after 0, 24, 48 and 72 hrs. Colonyforming units (CFUs) were counted after 4 days of incubation at 30°C. Results from each drug concentration are representative of at least 2 independent experiments. Cytotoxicity assay. THP-1 cells were differentiated with PMA for 48 hrs and exposed to decreasing concentrations of either RFB or RIF (starting at 200 µg/mL) for an additional 72 hrs at 37°C with 5% CO 2 . Following incubation, 10% (vol/vol) resazurin dye was added to each well and left to incubate for 4 hrs at 37°C and 5% CO 2 . Data was acquired using a fluorescent plate reader (excitation 540 nm, emission 590 nm). DMSO was included as a negative control, while SDS was included as a positive control.

Intracellular killing assay. Human THP-1 monocytes were grown in RPMI medium supplemented with 10% Fetal bovine serum (Sigma Aldrich) (RPMI FBS ) and incubated at 37°C in the presence of 5% CO 2 .

Cells were differentiated into macrophages in the presence of 20 ng/mL Phorbol Myristate Acetate (PMA) in 24-well flat-bottom tissue culture microplates (10 5 cells/mL) and incubated for 48 hrs at 37°C with 5% CO 2 . Infection with clinical isolates or M. abscessus harbouring pTEC27 fluorescent tdTomato was carried out at 37°C in the presence of 5% CO 2 for 3 hrs at a MOI 2:1. After extensive washing with 1X phosphate buffered saline (PBS), cells were incubated with RPMI FBS containing 250 µg/mL amikacin for 2 hrs and washed again with PBS prior to the addition of 500 µL RPMI FBS containing DMSO (negative control) or 500 µL RPMI FBS containing 50 µg/mL of RIF or AMK, or 12.5 µg/mL of RFB. Macrophages were washed with PBS and lysed with 100 µL of 1% Triton X-100 at required time points. Serial dilutions of macrophage lysates were plated onto LB agar plates and colonies were counted to determine intracellular CFUs.

Microscopy-based infectivity assays. Monocytes were differentiated into macrophages (THP-1) in the presence of PMA and were grown on coverslips in 24-well plates at a density of 10 5 cells/mL for 48 hrs at 37°C with 5% CO 2 prior to infection with Tdtomato expressing M. abscessus for 3 hrs at a MOI of 2:1. After washing and AMK treatment to remove the extracellular bacilli, macrophages were exposed to DMSO (negative control), or 50 µg/mL RIF or AMK, or 12.5 µg/mL RFB, and fixed at 0, 1 and 3 days post-infection with 4% paraformaldehyde in PBS for 20 min. Cells were then permeabilized using 0.2% Triton X-100 for 20 min, blocked with 2% BSA in PBS supplemented with 0.2% Triton X-100 for 20 min, incubated with anti-CD63 antibodies (Becton Dickinson); dilution 1:1000) for 1 hr and with an Alexa Fluor 488-conjugated anti-mouse secondary antibody (Molecular Probes, Invitrogen). After 5 min of incubation with DAPI (dilution 1:1000), cells were mounted onto microscope slides using Immu-mount (Calbiochem) and examined with an epifluorescence microscope using a 63X objective. The average proportion of macrophages containing fewer than <5, 5-10, or >10 bacilli were quantified using Zeiss Axio-vision software. Images were acquired by focusing on combined signals (CD63 in green and red fluorescent M. abscessus) and captured on a Zeiss Axio-imager confocal microscope equipped with a 63X oil objective and processed using Zeiss Axiovision software. Quantification and scoring of the numbers of bacilli present within macrophages were performed using ImageJ. Equal parameters for the capture and scoring of images were consistently applied to all samples. For each condition, approximately 1000 infected macrophages were analyzed. The presence of the intra-or extracellular cords within or among the macrophages infected with the R morphotype strain were treated in the presence of DMSO, RIF, RFB or AMK at the concentrations previously described, counted and imaged using confocal microscopy.

Assessment of RFB efficacy in infected zebrafish. Experiments in zebrafish were conducted according to the Comité d'Ethique pour l'Expérimentation Animale de la Région Languedoc Roussillon under the reference CEEALR36-1145. Experiments were performed using the golden mutant [START_REF] Lamason | SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans[END_REF]. Embryos were obtained and maintained as described [START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF]. Embryo age is expressed as hours post fertilisation (hpf).

Red fluorescent M. abscessus CIP104536 T (R) expressing tdTomato were prepared and microinjected in the caudal vein (2-3 nL containing ≈100 bacteria/nL) in 30 hpf embryos previously dechorionated and anesthetized with tricaine, as described earlier [START_REF] Bernut | Deciphering and imaging pathogenesis and cording of Mycobacterium abscessus in zebrafish embryos[END_REF]. The bacterial inoculum was checked a posteriori by injection of 2 nL in sterile PBS T and plating on 7H10 OADC . Infected embryos were transferred into 24-well plates (2 embryos/well) and incubated at 28.5°C to monitor kinetics of infection and embryo survival. Survival curves were determined by counting dead larvae daily for up to 12 days, with the experiment concluded when uninfected embryos started to die. RFB treatment of infected embryos and uninfected embryos was commenced at 24 hpi (hours post-infection) for 4 days. The drug-containing solution was renewed daily. Bacterial loads in live embryos were determined by anesthetising embryos in tricaine as previously described [START_REF] Bernut | In vivo assessment of drug efficacy against Mycobacterium abscessus using the embryonic zebrafish test system[END_REF], mounting on 3% (w/v) methylcellulose solution and taking fluorescent images using a Zeiss Axio Zoom.V16 coupled with an Axiocam 503 mono (Zeiss). Fluorescence Pixel Count (FPC) measurements were determined using the 'Analyse particles' function in ImageJ [START_REF] Bernut | Deciphering and imaging pathogenesis and cording of Mycobacterium abscessus in zebrafish embryos[END_REF]. Bacterial cords were identified based on the size and shape of fluorescent bacteria within the live zebrafish embryo, vastly exceeding the surrounding size and shape of neighbouring cells. All experiments were completed at least three times independently. Western blotting. Bacteria were harvested, resuspended in PBS, and disrupted by bead-beating with 1-mm diameter glass beads. The protein concentration in the lysates was determined and equal amounts of proteins (100 µg) were subjected to SDS/PAGE. Proteins were transferred to a nitrocellulose membrane. For detection of Tap-HA and KasA (loading control), the membranes were incubated for 1 hr with either the rat anti-HA or rat anti-KasA antibodies (dilution 1:2000), washed, and subsequently incubated with goat anti-rat antibodies conjugated to HRP (Abcam, dilution 1:5000). The signal was revealed using the ChemiDoc MP system (Bio-Rad).

Overexpression of

RESULTS

Rough M. abscessus is more susceptible to RFB treatment than smooth M. abscessus in vitro.

Exposure of exponentially-growing M. abscessus CIP104536 T S and R isogenic variants to increasing concentrations of RFB, starting at 25 µg/mL for S and 6.25 µg/mL for R, resulted in a noticeable growth inhibition (Fig. 1). At the lowest concentration, the CFUs at 72 hrs post-treatment remained comparable to those of the inoculum, suggestive of a bacteriostatic effect. However, the highest RFB concentrations for both S (200 µg/mL) and R (50 µg/mL) variants were accompanied by 1.81 and 2.47

Log reduction in the CFU counts at 72 hrs post-treatment, respectively (Fig. 1). While similar bactericidal effects of RFB were observed against both variants, this was achieved with lower concentrations of RFB against the R variant relative to the isogenic S variant. Overall, RFB at concentratios of 12.50 µg/mL (For R) resulted in a killing effect comparable to the one of imipenem (IPM) used at the MIC (16 µg/mL), known as an active -lactam drug against M. abscessus (41) (Fig. 1).

To confirm the differences in the susceptibility to RFB, we determined the MIC of the CIP104536 T S and R variants in CaMHB. Table 1 clearly shows that the S strain is 4-fold more resistant than its R counterpart. However, both variants were similarly resistant to other rifamycins (RIF, RPT and RFX), in agreement with previous studies [START_REF] Pryjma | Rifabutin acts in synergy and is bactericidal with frontline Mycobacterium abscessus antibiotics clarithromycin and tigecycline, suggesting a potent treatment combination[END_REF][START_REF] Dick | Rifabutin is active against Mycobacterium abscessus in mice[END_REF]. Our MIC values, obtained in repetitive experiments with two different commercial sources of RFB, were higher than those reported earlier [START_REF] Pryjma | Rifabutin acts in synergy and is bactericidal with frontline Mycobacterium abscessus antibiotics clarithromycin and tigecycline, suggesting a potent treatment combination[END_REF][START_REF] Dick | Rifabutin is active against Mycobacterium abscessus in mice[END_REF], but comparable to values reported in another study [START_REF] Story-Roller | Select β-lactam combinations exhibit synergy against Mycobacterium abscessus in vitro[END_REF]. Consistently with other studies [START_REF] Aziz | Rifabutin is active against Mycobacterium abscessus complex[END_REF], we also noticed that the MIC values were dependent on the culture medium (Table S1). Interestingly, MICs of RFB against S and R strains were lower in Middlebrook 7H9 as compared to CaMHB but this effect was lost when supplementing the medium with OADC enrichment. In contrast the S and R strains displayed equal susceptibility levels to RFB in Sauton's medium.

To investigate the relationship between RFB activity and GPL production, drug susceptibility was assessed in CaMHB using the GPL-deficient ΔmmpL4b mutant, generated in the S background of the type strain CIP104536 T , and its complemented counterpart [START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF][START_REF] Medjahed | Construction of Mycobacterium abscessus defined glycopeptidolipid mutants: comparison of genetic tools[END_REF][START_REF] Roux | Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants[END_REF]. The mmpL4b gene encodes the MmpL4b transporter which participates in the translocation of GPL across the inner membrane [START_REF] Medjahed | Construction of Mycobacterium abscessus defined glycopeptidolipid mutants: comparison of genetic tools[END_REF][START_REF] Ripoll | Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus[END_REF]. Mutations in this gene are associated with loss of GPL and acquisition of a R morphotype [START_REF] Nessar | Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response[END_REF][START_REF] Medjahed | Construction of Mycobacterium abscessus defined glycopeptidolipid mutants: comparison of genetic tools[END_REF]. The parental S strain and, to a lesser extent the ΔmmpL4b-complemented strain, showed reduced susceptibility to RFB (MIC 32-64 µg/mL) than the M. abscessus R strain and the GPL-deficient ΔmmpL4b mutant (MIC 16 µg/mL) (Table 1). The MIC results are in agreement with the growth inhibition kinetics (Fig. 1) and suggest that the outer GPL layer influences the activity of RFB.

M. abscessus possesses numerous potential drug efflux systems [START_REF] Ripoll | Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus[END_REF], including MAB_1409c, a homolog of Rv1258c, previouly reported to mediate efflux of RIF in M. tuberculosis [START_REF] Balganesh | Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates[END_REF]. We thus addressed whether overexpression of MAB_1409c induces resistance to RFB in M. abscessus.

MAB_1409c was cloned in frame with a HA-tag in the integrative pMV306. The resulting construct pMV306-MAB_1409c-HA was introduced in both S and R variants and the expression of MAB_1409c was confirmed by Western blot analysis using anti-HA antibodies (Fig. S1). Drug susceptibility assessment indicated a 4-fold upshift in the MIC of RFB against the R strain carrying pMV306-MAB_1409c-HA while no changes in the MIC were observed with the S strain overproducing MAB_1409c (Table 1). This suggests that increasing expression of MAB_1409c in the R variant is likely to mediate efflux of RFB, leading to reduced susceptibility to the drug.

RFB is active against S and R M. abscessus isolates in vitro.

The activity of RFB was next tested using a set of clinical strains isolated from CF patients or non-CF patients. In general, the MIC of R strains were 2 to 4 times lower than those of S strains, altough there were variations among the strains (Table 2). Some R strains (10, 112, 179, 210) exhibited higher MIC values (100 µg/mL) than the reference CIP104536 T R strain, while one S strain appeared particularly susceptible to RFB (M.

massiliense 120 with a MIC of 6.25 µg/mL). These differences between strains and S/R morphotypes were not observed previously with BDQ and S and R variants were also equally sensitive to BDQ [START_REF] Dupont | Bedaquiline inhibits the ATP synthase in Mycobacterium abscessus and is effective in infected zebrafish[END_REF].

Overall, these results demonstrate that RFB is active against M. abscessus, including isolates from CF patients, while R variants appear in general more susceptible to RFB than S variants, supporting previous findings [START_REF] Cheng | In vitro synergism of rifabutin with clarithromycin, imipenem, and tigecycline against the Mycobacterium abscessus complex[END_REF].

Mutations in rpoB confer resistance to RFB. Although rifamycin resistance mechanisms mediated by mutations in the rpoB gene coding for the -subunit of RNA polymerase have been widely described for M. tuberculosis, this is not the case for M. abscessus. Therefore, to identify the mechanism of resistance of RFB, a genetic approach involving the selection of spontaneous RFB-resistant mutants of M. abscessus followed by rpoB sequencing was applied. Four spontaneous strains were isolated in the presence of 25 or 50 µg/mL RFB, exhibiting 4-to 8-fold increased resistance levels as compared to the parental strain, respectively (Table 3). Sequencing analyses of rpoB identified several single nucleotide polymorphisms (SNPs) across four resistor mutants. In mutants 25.1 and 50.1, a C1339T

substitution was identified at position 447 (H447Y). Similarly, in mutant 25.2, a C1339G replacement was found, resulting in an amino acid substitution at position 447 (H447D). Comparatively, in mutant strain 50.2, another SNP (C1355T) occurred, leading to an amino acid change at position 452 (S452L).

A comparison of growth of different resistant strains on agar plates containing increasing concentrations of RFB is shown in Fig. S2. Whereas RFB abrogated growth of the wild-type S and R strains, growth of all four resistors harbouring mutations at either positions 447 or 452 sustained bacterial growth at 50 µg/mL, confirming that mutations in rpoB confer resistance to RFB.

M. abscessus S and R strains are equally susceptible to RFB in macrophages. While RFB has been shown to be active against M. tuberculosis in a macrophage infection model ( 49), this has not been thoroughly investigated for M. abscessus. We thus compared the intracellular efficacy of RFB in THP-1 macrophages infected with either S or R variants. Firstly, the cytotoxicity of RFB and RIF against THP-1 cells was investigated over a 3-days exposure period to either drug. Fig. S3, clearly shows that RFB exerts significant cytotoxicity at concentration >25 µg/ml and that the kinetic of macrophage killing was more rapid with RFB than with RIF. Based on these results, all subsequent macrophage studies were treated with 50 µg/mL RIF or 12.5 µg/mL RFB. AMK at 50 µg/mL was added as a positive control. DMSO-treated macrophages were included as a negative control for intracellular bacterial replication. At 0, 1 and 3 days post-infection (dpi), macrophages were lysed and plated to determine the intracellular bacterial loads following drug treatment. Whereas the presence of DMSO or RIF failed to inhibit intramacrophage growth of M. abscessus S, exposure to RFB strongly decreased the intracellular bacterial loads at 1 dpi, with this effect further exacerbated at 3 dpi (Fig. 2A). As anticipated, treatment with RIF did not show any effect, in agreement with the poor activity of this compounds in vitro (Table 1). Comparatively, AMK treatment resulted in a significantly reduced intracellular growth rate in both M. abscessus S and R variants between 1 and 3dpi. Interestingly, the a ~3 Log reduction in the CFU counts (Fig. 2A andB, respectively).

Macrophages were next infected with M. abscessus strains expressing Tdtomato and exposed to either DMSO, AMK, RIF or RFB, followed by staining with anti-CD63 and DAPI and observed under a confocal microscope. A quantitative analysis confirmed the marked reduction in the number of M.

abscessus S-infected THP-1 cells treated with AMK and RFB at 1 and 3 dpi compared to RIF-treated cells or untreated control cells (Fig. 2C). A similar trend was observed when macrophages were infected with M. abscessus R (Fig. 2D).

Macrophages infected with the S variant were then classified into three categories based on their bacterial burden: poorly infected (<5 bacilli), moderately infected (5-10 bacilli) and heavily infected (>10 bacilli) macrophages. Cells containing bacilli were then individually observed under the microscope and scored to one of the three categories. The quantitative analysis indicates that exposure to RFB significantly reduces the percentage of S variant heavily infected THP-1 cells while increasing the proportion of the poorly infected category, as compared to the untreated cells at 1 dpi (Fig. 2E). At 3 dpi, the effect of RFB was even more pronounced with 10% of the infected bacilli belonging to the heavily infected category and more than 50% associated with the poorly infected category. Analysis performed on cells infected with the R variant generated a similar category profile, although treatment with RFB was associated with a higher proportion of heavily infected macrophages at 3 dpi with the R variant than with the S variant (Fig. 2F). Fig. 2G illustrates the reduced number of M. abscessus S in infected THP-1 cells treated with RFB at 1 dpi, as compared to the untreated control cells (DMSO) or those treated with RIF or AMK. Collectively, these results

indicate that RFB enters THP-1 macrophages and similarly impedes bacterial replication of both M. abscessus S and R variants.

RFB reduces the intramacrophage growth of clinical isolates. RFB has recently shown vast potential

as an effective antibiotic for the treatment of M. abscessus infection in a NOD/SCID murine model [START_REF] Dick | Rifabutin is active against Mycobacterium abscessus in mice[END_REF]. However, to date the efficacy of RFB has only been evaluated against a limited panel of M.

abscessus clinical isolates within an infection setting. As such, we explored the activity of RFB against S and R clinical isolates of the M. abscessus complex with varying MIC values against RFB within THP-1 macrophages. In support of our previous findings in infected macrophages, RFB treatment (12.5 or 25 µg/mL) was very active against all M. abscessus subspecies within macrophages at 1 and 3 dpi when compared to Day 0 and DMSO treatment (Fig. 3), irrespective of S and R morphotypes and the corresponding MIC values (Table 2). Overall, these findings suggest that RFB is very effective against intracellular clinical isolates and highlights the lack of direct correlation between MICs determined in vitro and the intracellular activity of RFB.

Reduced intra-and extracellular cording by RFB treatment. An important phenotypic difference between S and R morphotypes is that R morphotypes display increased bacterial aggregation. R bacilli remain attached during replication, forming compact colonies containing structures that resemble cords on agar and in broth medium [START_REF] Howard | Spontaneous reversion of Mycobacterium abscessus from a smooth to a rough morphotype is associated with reduced expression of glycopeptidolipid and reacquisition of an invasive phenotype[END_REF][START_REF] Sánchez-Chardi | Demonstration of cord formation by rough Mycobacterium abscessus variants: implications for the clinical microbiology laboratory[END_REF][START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF]. Fig. 4A clearly shows that, upon infection with M.

abscessus R expressing TdTomato, the total number of cords per field was significantly reduced in the presence of 50 µg/mL AMK or 12.5 µg/mL RFB when compared to 50 µg/mL RIF or DMSO alone.

Moreover, we observed intracellular cords that are capable of growing inside the macrophage as well as in the extracellular milieu, which were easily observable at 3 dpi (Fig. 4B). As illustrated in Fig. 4C, treatment with AMK or RFB strongly impacted on both intra-and extracellular cords. While AMK treatment severely reduced the number of both intra-and extracellular cords, this effect was almost completely abrogated with RFB at 3 dpi. Together, these results indicate that RFB is highly effective in reducing M. abscessus cords, thought to affect the outcome of the infection.

RFB treatment enhances protection of zebrafish infected with M. abscessus.

In vivo drug efficacy has previously been well described using the zebrafish model of infection [START_REF] Bernut | In vivo assessment of drug efficacy against Mycobacterium abscessus using the embryonic zebrafish test system[END_REF][START_REF] Dupont | Bedaquiline inhibits the ATP synthase in Mycobacterium abscessus and is effective in infected zebrafish[END_REF][START_REF] Dubée | β-Lactamase inhibition by avibactam in Mycobacterium abscessus[END_REF]. Initial experiments indicated that RFB concentrations ≤ 100 µg/mL (final concentration in fish water) did not interfere with larval development and was well tolerated in embryos when treatment was applied for 4 days with daily drug renewal (Fig. 5A). Higher concentrations of RFB, however, were associated with rapid larval death. As such, only lower RFB doses (≤ 100 µg/mL) were used in subsequent studies. Red fluorescent tdTomato-expressing M. abscessus (R variant) was microinjected in the caudal vein of embryos at 30 hrs post-fertilisation (hpf). RFB was directly added at 1 dpi to the water containing the infected embryos, with RFB-supplemented water changed on a daily basis for 4 days.

Embryo survival was monitored and recorded daily for 12 days. No decrease in the survival rate was observed in the presence of 5 µg/mL RFB, however, a significant dose-dependent increase in the survival of embryos exposed to 25 or 50 µg/mL RFB was observed as compared to the untreated group (Fig. 5B). When exposed to 50 µg/mL RFB, the highest dose examined in this setting, nearly 80% of the treated embryos survived at 12 dpi, as compared to 40% of the untreated group. This clearly indicates that RFB protects zebrafish from M. abscessus infection.

To test whether RFB exerts an effect on the bacterial burden in zebrafish, we quantified fluorescent pixel counts (FPC) [START_REF] Bernut | Deciphering and imaging pathogenesis and cording of Mycobacterium abscessus in zebrafish embryos[END_REF]. As expected, embryos treated with 50 µg/mL RFB had significantly decreased bacterial burdens at 2, 4 and 6 dpi when compared to the untreated group (Fig. 5C). These results were corroborated by imaging whole embryos, characterised by the presence of large abscesses and cords in the brain when left untreated and which were observed much less frequently in the RFB-treated animals despite the presence of single bacilli or small aggregated bacteria (Fig. 5D).

RFB treatment reduces abscess formation by M. abscessus in zebrafish. Virulence of M. abscessus R variants in zebrafish are correlated with the presence of abscesses, particularly in the central nervous system [START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF][START_REF] Bernut | Deciphering and imaging pathogenesis and cording of Mycobacterium abscessus in zebrafish embryos[END_REF]. To address whether the enhanced survival of RFB-treated fish is associated with decreased abscess formation, the percentage of abscesses and cords were determined by monitoring abscesses and cords in whole embryos, as reported previously [START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF][START_REF] Bernut | Deciphering and imaging pathogenesis and cording of Mycobacterium abscessus in zebrafish embryos[END_REF]. Extracellular cords can be easily distinguished based on their serpentine-like shape and by their size, often greater as compared to the size of the surrounding macrophages and neutrophils. Exposure of infected embryos to 50 µg/mL RFB was accompanied by a significant decrease in the proportion of embryos with cords (Fig. 6A) at 4 dpi, and the number of embryos with abscesses (Fig. 6B) at 4 and 6 dpi. This decrease in the physiopathological signs of RFB-treated larvae correlates also with the FPC analysis and whole embryo imaging (Fig. 6C and6D). Overall, these results demonstrate that RFB reduces the pathophysiology of M. abscessus infection in zebrafish larvae and protects them from bacterial killing.

DISCUSSION

Treatment success of infections caused by M. abscessus is unacceptably low even upon prolonged, multidrug chemotherapy with a significant risk of severe toxic side effects. Although RIF is used as a first-line drug against M. tuberculosis, it has no activity against M. abscessus. While ADP ribosyltransferases can utilise both RIF and RFB as substrates [START_REF] Baysarowich | Rifamycin antibiotic resistance by ADP-ribosylation: Structure and diversity of Arr[END_REF], a lower catalytic efficiency with RFB may explain its greater potency against M. abscessus. Our study supports and extends previous investigations highlighting the potential of RFB against M. abscessus in vitro against a wide panel of M. abscessus complex clinical isolates [START_REF] Aziz | Rifabutin is active against Mycobacterium abscessus complex[END_REF][START_REF] Pryjma | Rifabutin acts in synergy and is bactericidal with frontline Mycobacterium abscessus antibiotics clarithromycin and tigecycline, suggesting a potent treatment combination[END_REF][START_REF] Ganapathy | Repositioning rifamycins for Mycobacterium abscessus lung disease[END_REF][START_REF] Run | In vitro and intracellular activity of imipenem combined with rifabutin and avibactam against Mycobacterium abscessus[END_REF]. We found, however, that our MIC values were higher than those observed in previous investigations [START_REF] Aziz | Rifabutin is active against Mycobacterium abscessus complex[END_REF][START_REF] Pryjma | Rifabutin acts in synergy and is bactericidal with frontline Mycobacterium abscessus antibiotics clarithromycin and tigecycline, suggesting a potent treatment combination[END_REF]. In our study, following the Clinical and Laboratory Standard Institute (CLSI) guidelines, MIC were determined in CaMHB while Aziz et al.

showed that MIC values were 2-to 3-fold higher in CaMHB as compared to Middlebrook 7H9 [START_REF] Aziz | Rifabutin is active against Mycobacterium abscessus complex[END_REF],

clearly implicating an effect of medium on RFB susceptibility testing. In line with these results, we noticed important variations in the MIC values depending on the culture medium used for RFB susceptibility assessments. It is also noteworthy that the growth curve of the untreated S strain is different from the one of the R strain, which is very likely linked to the highly aggregative surface properties typifying the R strain which, in contrast to the S strain, produces very clumpy and corded cultures in broth medium [START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF][START_REF] Bernut | Deciphering and imaging pathogenesis and cording of Mycobacterium abscessus in zebrafish embryos[END_REF][START_REF] Brambilla | Mycobacteria clumping increase their capacity to damage macrophages[END_REF]. As a consequence, colonies on agar plates are very likely emerging from aggregated bacteria rather than individual bacilli, explaining why the CFU counts were significantly lower in both cultures. Thus, the CFU counts of the R strain does not accurately reflect the absolute number of living bacilli in the culture. We also selected RFB-resistant mutants and identified mutations in rpoB, known as the primary target of rifampicin in M. tuberculosis [START_REF] Jagielski | Characterization of mutations conferring resistance to rifampin in Mycobacterium tuberculosis clinical strains[END_REF].

Interestingly, the mutations identified are part of the rifampicin-resistance-determining region (RRDR), a 81-bp central segment corresponding to codons 426 to 452 in M. tuberculsosis that harbours the vast majority of rpoB mutations associated with resistance to RIF [START_REF] Jagielski | Characterization of mutations conferring resistance to rifampin in Mycobacterium tuberculosis clinical strains[END_REF]. Noteworthingly, S452L corresponds to one of the most frequently mutated coding region in the rpoB gene in M.

tuberculosis (S450L replacement) [START_REF] Jagielski | Characterization of mutations conferring resistance to rifampin in Mycobacterium tuberculosis clinical strains[END_REF]. Together, these results suggest RpoB is very likely the target of RFB in M. abscessus.

Among the various studies reporting the activity of RFB against M. abscessus in vitro, very few discriminated the activity of RFB against the S or R morphotypes. Herein, we found that the type strain CIP104536 T S was reproducibly more resistant to RFB than its R counterpart. Supporting these results, deletion of mmpL4b in the S genetic background, resulting into an R morphotype lacking GPL [START_REF] Nessar | Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response[END_REF][START_REF] Medjahed | Construction of Mycobacterium abscessus defined glycopeptidolipid mutants: comparison of genetic tools[END_REF], increased susceptibility to RFB. Conversely, functional complementation of the mmpL4b mutant, restoring the S morphotype and GPL production [START_REF] Nessar | Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response[END_REF][START_REF] Medjahed | Construction of Mycobacterium abscessus defined glycopeptidolipid mutants: comparison of genetic tools[END_REF], partially rescued the higher MIC.

This highlights the influence of the outermost GPL layer on susceptility to RFB. Previously, the activity of other inhibitors have been shown to be dependent on the presence or absence of GPL in M.

abscessus [START_REF] Madani | Cyclipostins and Cyclophostin analogues as multitarget inhibitors that impair growth of Mycobacterium abscessus[END_REF][START_REF] Lavollay | In vitro activity of cefoxitin and imipenem against Mycobacterium abscessus complex[END_REF]. A logical explanation is that the GPL layer protects the bacilli from the penetration of drugs. The absence of GPL may enhance the permeability of the cell wall and accumulation of the drug inside the bacteria. However, one cannot exclude the possibility that MmpL4b, like other MmpL transporters, can act as an efflux pump (56-58) and may participate in the extrusion of RFB in M. abscessus S, resulting in higher MIC. The implication of efflux pumps in resistance to RFB has been investigated, whereby the overexpression of MAB_1409c (a homologue of the M. tuberculosis Rv1258c) resulted in increased resistance to RFB in the R variant of M. abscessus. This effect was not observed in the S strain overexpressing MAB_1409c, presumably because of the already elevated MIC of the parental S strain towards RFB. However, while the increased susceptibility of the R strain as compared to the S strain was true with respect to the type strain, this was not observed for all clinical strains tested. The heterogeneity of the clinical strains in response to RFB treatment cannot be simply explained by the presence or absence of GPL, but may also include additional determinants of resistance to RFB [START_REF] Ganapathy | Repositioning rifamycins for Mycobacterium abscessus lung disease[END_REF], such as differences in the expression level of Arr Mab or the expression of Rox monooxygenases, known to inactivate RIF in other bacterial species as proposed earlier [START_REF] Koteva | Rox, a rifamycin resistance enzyme with an unprecedented mechanism of action[END_REF]. This, however, requires further investigation in follow-up studies.

One unanticipated finding from this study relies on the fact that, although S and R variants respond differently to RFB treatment in vitro, this was not the case against the intracellularly-residing M. abscessus. We found that, using a macrophage model of infection, the isogenic S and R type strains responded equally well to treatment with 12.5 µg/mL RFB, largely exceeding the results obtained with AMK, a drug displaying weak intracellular activity [START_REF] Lefebvre | Inhibition of the β-lactamase BlaMab by avibactam improves the in vitro and in vivo efficacy of imipenem against Mycobacterium abscessus[END_REF]. These observations are reminiscent of other studies indicating that various naphtalenic ansamycins, including RIF, differ profoundly in their capacity to kill extracellular Staphylococcus aureus, albeit there were few differences observed between them in promoting human macrophages to kill phagocytosed bacteria [START_REF] Marshall | MIC values do not predict the intraphagocytic killing of Staphylococcus aureus by naphthalenic ansamycins[END_REF]. There is no simple explanation as why M. abscessus S is as efficiently killed as M. abscessus R inside the cells. A plausible explanation may be that the stress response inside macrophages alters the composition/architecture of the cell wall of M. abscessus, thereby affecting the GPL layer and/or permeability of the S variant. It has been shown that the GPL layer significantly influences the hydrophobic surface properties [START_REF] Viljoen | Fast chemical force microscopy demonstrates that glycopeptidolipids define nanodomains of varying hydrophobicity on mycobacteria[END_REF], potentially impacting on the adhesion and the uptake of the bacilli. Furthermore, electron microscopy observations revealed that the electron translucent zone (ETZ) that fills the entire space between the phagosome and the bacterial surface relies on GPL production in the S variant [START_REF] Bernut | Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members[END_REF]. Alternatively, RFB may directly induce the antimycobacterial activity of the macrophage, which in turns translates into a rapid killing of the phagocytosed bacteria, regardless of their morphotype. Overall, these results suggest that the MIC values of RFB are not indicative of the intraphagocytic killing of M. abscessus and highlights the importance of testing the efficacy of drugs in a macrophage infection model.

Cords and abscesses are pathophysiological markers of M. abscessus infection, as revealed using the zebrafish model of infection [START_REF] Bernut | In vivo assessment of drug efficacy against Mycobacterium abscessus using the embryonic zebrafish test system[END_REF]. In particular, extracellular cords, due to their size, prevent the bacilli from being phagocytosed by macrophages and neutrophils, representing an important mechanism of immune evasion [START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF][START_REF] Bernut | Deciphering and imaging pathogenesis and cording of Mycobacterium abscessus in zebrafish embryos[END_REF]. We demonstrate here that treatment of infected macrophages was associated with reduced intra-and extracellular cording of the R variant. It is very likely that RFB prevents cording, as a consequence of the inhibition of bacterial replication/killing.

Cords are a hallmark of virulence of the R variant of M. abscessus, as emphasized by a deletion mutant of MAB_4780, encoding a dehydratase, displaying a pronounced defect in cording and a highly attenuated phenotype in macrophages [START_REF] Halloum | Deletion of a dehydratase important for intracellular growth and cording renders rough Mycobacterium abscessus avirulent[END_REF]. Importantly, we observed also a significant decrease in the number of embryos with cords following RFB treatment in infected zebrafish. It is worth highlighting that in the presence of RFB, there is no change in the number of embryos with cords between 2 and 4 dpi, implying that while RFB does not degrade or modify the bacterial cord structure, it likely prevents the formation of additional cords. Moreover, the effect of RFB on cord reduction is particularly interesting as it may prevent the subsequent formation of abscesses [START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF], considered as a marker of severity of the disease. Consistent with this hypothesis, a marked decrease in abscess formation was observed in RFB-treated zebrafish embryos. Overall, this work supports the practicality of zebrafish as a pre-clinical model to evaluate in real-time the bactericidal efficacy of RFB against M. abscessus infection in the sole context of innate immunity.

In summary, although there is a clear lack of bactericidal activity of drugs against M. abscessus (64), these findings support the high activity of RFB against M. abscessus in vivo and in vitro. Our results further emphasize the efficacy of RFB against both extracellular and intracellular forms of M.

abscessus, both co-existing in infected patients, as well as a protective effect in an animal model of M. abscessus infection. In addition, we have provided further evidence that S and R variants are differentially susceptible to RFB, likely due to the GPL layer, however the MIC values are not predictive of intracellular drug efficacy.

Together with the fact that RFB is an FDA-approved drug that is already used to treat tuberculosis [START_REF] Lee | Treatment outcomes of rifabutin-containing regimens for rifabutin-sensitive multidrug-resistant pulmonary tuberculosis[END_REF] and M. avium infections [START_REF] Griffith | Treatment of Mycobacterium avium complex (MAC)[END_REF] with favourable pharmacological properties [START_REF] Blaschke | The clinical pharmacokinetics of rifabutin[END_REF], our data strengthen the view that RFB should be considered as a repurposing drug candidate for the treatment of M. abscessus infections. Importantly, recent work has shown that RFB is synergistic in combinations with other antimicrobials such as clarithromycin, imipenem and tigecycline, and significantly improves the activity of imipenem-tedizolid drug combinations [START_REF] Pryjma | Rifabutin acts in synergy and is bactericidal with frontline Mycobacterium abscessus antibiotics clarithromycin and tigecycline, suggesting a potent treatment combination[END_REF][START_REF] Cheng | In vitro synergism of rifabutin with clarithromycin, imipenem, and tigecycline against the Mycobacterium abscessus complex[END_REF][START_REF] Run | In vitro and intracellular activity of imipenem combined with rifabutin and avibactam against Mycobacterium abscessus[END_REF][START_REF] Run | In vitro and intracellular activity of imipenem combined with tedizolid, rifabutin, and avibactam against Mycobacterium abscessus[END_REF]. Future studies are required to test whether these RFB combinations are effective against M. abscessus pulmonary infections.

  MAB_1409c in M. abscessus.Overexpression was achieved by PCR amplification of MAB_1409c (tap) in fusion with an HA tag using genomic DNA and the forward primer (5′-gagaCAATTGCCATGTCCACTCCGACGGCGGATTC-3′; MfeI) ; HpaI). The amplified product was digested with MfeI/HpaI and ligated into the MfeI/HpaI-restricted pMV306 integrative vector to generate pMV306-MAB_1409c-HA where MAB_1409c-HA is under the control of the hsp60 promoter. The construct was sequenced and electroporated in M. abscessus S and R.Selection of resistant M. abscessus mutants and target identification. Exponentially growing M. abscessus CIP104536 T R cultures were plated on LB agar containing either 25 or 50 µg/mL RFB. After one week of incubation at 37°C, four individual colonies from each RFB concentration were selected, grown in CaMHB, individually assessed for MIC determination and scored for resistance to RFB. Identification of SNPs in the resistant strains was completed by PCR amplification using rpoB_f 5'-TCAGTGGGGCTGGTTAG -3' and rpoB_r 5'-AAAACATCGCAGATGCGC-3' to produce a 3541 bp amplicon for full coverage sequencing of the rpoB gene.

Statistical analyses.

  Statistical analyses were performed on Prism 5.0 (Graphpad) and detailed for each figure legend. *P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Figure 1 .

 1 Figure 1. In vitro activity of rifabutin. M. abscessus CIP104536 T S (left panel) or R (right panel) was exposed either to 200, 100, 50, 25, 12.5 or 6.25 µg/mL RFB or 16 µg/mL IPM in CaMHB at 30°C. At various time points, bacteria were plated on LB agar and further incubated at 30°C for 4 days prior to CFU counting. Results are expressed as the mean of triplicates ± SD and are representative of two independent experiments. *P ≤ 0.05, ** P ≤ 0.01.

Figure 2 .

 2 Figure 2. Intracellular activity of RFB on M. abscessus-infected THP-1 cells. (A) Macrophages were infected with M. abscessus S-morphotype and (B) R-morphotype expressing tdTomato (MOI of 2:1) for 3 hrs prior to treatment with RIF (50 µg/mL), AMK (50 µg/mL), RFB (12.5 µg/mL) or DMSO. CFU were determined at 0, 1 and 3 dpi. Data are mean values ± SD for three independent experiments. Data were analysed using a one-way ANOVA Kruskal-Wallis test. (C) Percentage of infected THP-1 macrophages at 0, 1 and 3 days post-infection after infection with M. abscessus S or (D) M. abscessus R. Data are mean values ± SD for three independent experiments. Data were analysed using a oneway ANOVA Kruskal-Wallis test. (E) Percentage of S-infected macrophage categories and (F) percentage of R-infected macrophage categories infected with different numbers of bacilli (<5 bacilli; 5-10 bacilli and >10 bacilli). The categories were counted at 0 or at 1 and 3 days post-infection in the absence of antibiotics or in the presence of RIF or AMK at 50 µg/mL, or RFB at 12.5 µg/mL. Values are means ± SD from three independent experiments performed in triplicate. (G) Four immunofluorescent fields were taken at 1 day post-infection showing macrophages infected with M. abscessus expressing Tdtomato (red). The surface and the endolysosomal system of the macrophages were detected using anti-CD63 antibodies (green). The nuclei were stained with DAPI (blue). White arrows indicate individual or aggregate mycobacteria. Scale bar, 20 µm. ** P ≤ 0.01, *** P ≤ 0.001.

Figure 3 .

 3 Figure 3. Intracellular activity of RFB on S and R clinical isolates. CFU counts of clinical isolates exposed to 25 and 12.5 µg/mL RFB. Macrophages were infected with M. abscessus (A-B) M. bolletii (C-D) or M. massiliense (E-G) clinical strains belonging to S or R morphotypes at MOI of 2:1 for 3 hrs prior to treatment with 250 µg/mL AMK for 2 hrs to kill extracellular bacteria. Following extensive PBS washes, cells were exposed to 50 µg/mL RIF, 50 µg/mL AMK, 25 or 12.5 µg/mL RFB . CFU were

Figure 4 .

 4 Figure 4. Activity of RFB on extracellular and intracellular cords. (A) Total number of cords displayed in 20 fields at 3 days post-infection after infection of macrophages with M. abscessus R variant. Data are mean values ± SD for three independent experiments performed in triplicate. Data were analysed using one tailed Mann Whitney's t-test. (B) Percentage of cords formed either extracellularly or intracellularly. The two categories were counted at 3 days post-infection in the absence of antibiotics or in the presence of 50 µg/mL RIF, 50 µg/mL AMK or 12.5 µg/mL RFB. Extracellular or intracellular cords are highlighted using the indicated colour codes. Values are means ± SD for two independent experiments performed each time in triplicate. (C) Four immunofluorescent fields were taken at 3 days post-infection showing the cords formed extracellularly or within macrophages infected with M. abscessus R variant expressing Tdtomato (red). Macrophages were infected for 3 days in the presence of DMSO, RIF (50 µg/mL), AMK (50 µg/mL) or RFB (12.5 µg/mL). The macrophage surface was stained using anti-CD63 antibodies (green). The nuclei were stained with DAPI (blue). White arrows indicate intracellular cords, while red arrows indicate extracellular cords. Scale bars represent 20 µm. Results represent the average of a total of 120 fields per condition. **** P ≤ 0.0001.

Figure 5 .

 5 Figure 5. RFB displays high bactericidal activity against M. abscessus in an embryonic zebrafish infection model. (A) Groups of uninfected embryos were immersed in water containing increasing concentrations of RFB (ranging from 3.125 to 250 µg/mL) for 4 days. The red bar indicates the duration of treatment. The graph shows the survival of the RFB-treated and untreated (DMSO) embryos over a 12-days period. (B) Zebrafish embryos at 30 hrs post-fertilisation were intravenously infected with approximately 250-300 CFU of M. abscessus CIP104536 T (R variant) expressing tdTomato (n=20-25). A standard PBS injection control was included for each experiment. At 1 dpi, embryos were randomly split into equal groups of approximately 20 embryos per group, and varying concentrations of RFB (5 to 50 µg/mL) were added to the water. DMSO was included as a positive control group. RFB was changed daily after which, embryos were washed twice in fresh embryo water, maintained in embryo water and monitored daily over a 12-days period. Each treatment group

Table 1 .

 1 Drug susceptibility/resistance profile of smooth and rough variants derived from the reference M. abscessus 104536 T strain to various rifamycins in CaMBH. MIC (µg/mL) were determined following the CLSI guidelines.

					MIC (µg/mL)
	Strain	Morphotype	RFB	RIF	RPT	RFX
	CIP104536 (S)	S	64	>128	>128	>128
	CIP104536 (R)	R	16	>128	>128	>128
	ΔMAB_mmpL4b	R	16	>128	>128	>128
	ΔMAB_mmpL4b_C	S	32	>128	>128 >128
	CIP104536 (S) + pMV306-MAB_1409c-HA S	64	>128	>128 >128
	CIP104536 (R) + pMV306-MAB_1409c-HA R	64	>128	>128 >128

RFB, Rifabutin; RIF, rifampicin; RPT, rifapentine; RFX, rifaximin.

Table 2 . Comparison of the activity of RFB against clinical isolates from CF and non-CF patients.

 2 The 737 MIC (µg/mL) was determined in Cation-Adjusted Mueller-Hinton broth for different subspecies 738 belonging to the M. abscessus complex. Results are from 3 independent experiments. RFB, rifabutin.

	739
	740
	741 742 743 744 745 746	Strain M. abscessus CIP104536 3321 1298	Morphotype S S S	Source Non-CF Non-CF CF	RFB 50 50 50
		2587	S	CF
		2069	S	Non-CF
		CF	S	CF	25
		2524	R	CF	25
		2648	R	CF	25
		3022	R	Non-CF	50
		5175	R	CF	25
		CIP104536	R	Non-CF	25
		M. massiliense
		CIP108297	R	Addison Disease	50
		210	R	CF
		179	R	CF
		CIP108297	S	Addison Disease
		140	S	CF	50
		185	S	CF
		107	S	CF	50
		122	S	CF
		120	S	CF	6.25
		212	S	CF
		100	S	CF
		111	S	CF
		M. bolletii
		CIP108541	S	Non reported
		114	S	CF
		17	S	CF	50
		116	S	CF
		97	S	CF
		112	R	CF
		19	R	Non-CF	50
		10	R	Non reported
		108	R	CF	25

Table 3 . Characterisitics of spontaneous RFB-resistant mutants of M. abscessus

 3 . MIC (µg/mL) were determined in Cation-adjusted Mueller-Hinton broth. Resistant strains were derived from the rough M. abscessus CIP104536 T parental strain on Middlbrook 7H10 supplemented with either 25 or 50 µg/mL RFB. Single nucleotide polymorphism identification in rpoB (MAB_3869c) and corresponding amino acid changes are also indicated. RFB, rifabutin.

		MIC (µg/ml)	Mutation in rpoB
			SNP	AA change
	CIP104536 T (R)	12.5	-	-
	25.1	50	C1339T	H447Y
	25.2	100	C1339G	H447D
	50.1	50	C1339T	H447Y
	50.2	50	C1355T	S452L

ACKNOWLEDGMENTS

MDJ received a post-doctoral fellowship granted by Labex EpiGenMed, an «Investissements d'avenir» program (ANR-10-LABX-12-01). This study was supported by the Association Gregory Lemarchal and Vaincre la Mucoviscidose (RIF20180502320) to LK.

The authors have no conflict of interest to declare.

was compared against the untreated infected group with significant differences calculated using the log-rank (Mantel-Cox) statistical test for survival curves. Data shown is the merge of three independent experiments (C) Bacterial burden was determined at 2, 4 and 6 days post-infection following treatment with either DMSO or 50 µg/mL RFB. Bacteria were quantified by fluorescent pixel count determination using ImageJ software, with each data point representing a single embryo.