Mycobacterium abscessus HelR interacts with RNA polymerase to confer intrinsic rifamycin resistance.

Rifampicin (RIF), the frontline drug against M. tuberculosis, is completely ineffective against M. abscessus, partially due to the presence of an ADP-ribosyltransferase (Arr) that inactivates RIF. Using RNA-seq, we show that exposure of M. abscessus to sublethal doses of RIF and Rifabutin (RBT), a close analog of RIF, results in an ∼25-fold upregulation of Mab_helR in laboratory and clinical isolates. An isogenic deletion in Mab_helR results in RIF/RBT hypersensitivity, and overexpression of Mab_helR confers RIF tolerance in M. tuberculosis. We demonstrate an increased HelR-RNAP association in RIF-exposed bacteria and a MabHelR-mediated dissociation of RNAP from stalled initiation complexes in vitro. Finally, we show that the tip of the PCh-loop of Mab_helR, present in proximity to RIF, is critical for conferring RIF resistance but dispensable for dissociation of stalled RNAP complexes, suggesting that HelR-mediated RIF resistance requires a step in addition to displacement of RIF-stalled RNAP.

Mode of Action of Kanglemycin A, an Ansamycin Natural Product that Is Active against Rifampicin-Resistant Mycobacterium tuberculosis.

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered-compared with rifampicin-conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.

Genome-Driven Discovery of Hygrocins in Streptomyces rapamycinicus.

Ansamycins, represented by the antituberculosis drug rifamycin, are an important family of natural products. To obtain new ansamycins, Streptomyces rapamycinicus IMET 43975 harboring an ansamycin biosynthetic gene cluster was fermented in a 50 L scale, and subsequent purification work led to the isolation of five known and four new analogues, where hygrocin W (2) belongs to benzoquinonoid ansamycins, and the other three hygrocins, hygrocins X-Z (6-8), are new seco-hygrocins. The structures of ansamycins (1-8) were determined by the analysis of spectroscopic (1D/2D NMR and ECD) and MS spectrometric data. The Baeyer-Villiger enzyme which catalyzed the ester formation in the ansa-ring was confirmed through in vivo CRISPR base editing. The discovery of these compounds further enriches the structural diversity of ansamycins.

Rifabutin but not rifampicin can partly out-balance P-glycoprotein induction by concurrent P-glycoprotein inhibition through high affinity binding to the inhibitory site.

Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = -11.5 vs -5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp.

Antibiotic Adjuvant 4-Hexylresorcinol Enhances the Efficiency of Antituberculosis Drugs.

We studied the possibility of using 4-hexylresorcinol to increase the efficiency of anti-mycobacterial chemotherapy. In an in vitro experiment, 4-hexylresorcinol increased the efficiency of rifampicin, kanamycin, and isoniazid against Mycobacterium smegmatis by 3-5 times. Experiments in sanitation of BALB/c mice infected with M. smegmatis showed the best efficacy of the isoniazid and 4-hexylresorcinol combination in comparison with isoniazid monotherapy. The growth-inhibiting activity of the combination of antibiotic rifabutin with 4-hexylresorcinol was shown on 6 strains of M. tuberculosis. A 2-fold decrease in the minimum inhibitory concentration of this antibiotic in the presence of half-minimum inhibitory concentration of 4-hexylresorcinol was demonstrated for monoresistant strain M. tuberculosis 5360/42Hr. On the mouse model of experimental tuberculosis caused by M. tuberculosis H37Rv, a 5-fold decrease in lung contamination and more rapid complete cure were achieved in animals treated with the combination of rifabutin and 4-hexylresorcinol in comparison with rifabutin monotherapy.

ADP-ribosylation-resistant rifabutin analogs show improved bactericidal activity against drug-tolerant M. abscessus in caseum surrogate.

Necrotic lesions and cavities filled with caseum are a hallmark of mycobacterial pulmonary disease. Bronchocavitary Mycobacterium abscessus disease is associated with poor treatment outcomes. In caseum surrogate, M. abscessus entered an extended stationary phase showing tolerance to killing by most current antibiotics, suggesting that caseum persisters contribute to the poor performance of available treatments. Novel ADP-ribosylation-resistant rifabutin analogs exhibited bactericidal activity against these M. abscessus persisters at concentrations achievable by rifamycins in caseum.

In Vitro Synergistic Effects of Omadacycline with Other Antimicrobial Agents against Mycobacterium abscessus.

The clinical importance of Mycobacterium abscessus species (MABS) infections has been increasing. However, the standard treatment regimens recommended in the current guidelines often result in unfavorable outcomes. Therefore, we investigated the in vitro activity of omadacycline (OMC), a novel tetracycline, against MABS to explore its potential as a novel therapeutic option. The drug susceptibilities of 40 Mycobacterium abscessus subsp. abscessus (Mab) clinical strains obtained from the sputum of 40 patients from January 2005 to May 2014 were investigated. The MIC results for OMC, amikacin (AMK), clarithromycin (CLR), clofazimine (CLO), imipenem (IPM), rifabutin (RFB), and tedizolid (TZD) alone and their combined effects (with OMC) were examined using the checkerboard method. Additionally, we studied the differences in the effectiveness of the antibiotic combinations based on the colony morphotype of Mab. The MIC50 and MIC90 of OMC alone were 2 and 4 µg/mL, respectively. The combinations of OMC with AMK, CLR, CLO, IPM, RFB, and TZD showed synergy against 17.5%, 75.8%, 25.0%, 21.1%, 76.9%, and 34.4% of the strains, respectively. Additionally, OMC combined with CLO (47.1% versus 9.5%, P = 0.023) or TZD (60.0% versus 12.5%, P = 0.009) showed significantly higher synergy against strains with rough morphotypes than those with smooth morphotypes. In conclusion, the checkerboard analyses revealed that the synergistic effects of OMC were observed most frequently with RFB, followed by CLR, TZD, CLO, IPM, and AMK. Furthermore, OMC tended to be more effective against rough-morphotype Mab strains.

[In vitro Activity of Rifabutin and Clofazimine to Macrolide-Resistant Mycobacterium abscessus Complex Clinical Isolates]. / Rifabutin ve Klofaziminin Makrolidlere Dirençli Mycobacterium abscessus Kompleks Klinik Izolatlarina In vitro Etkinligi.

Mycobacterium abscessus complex (MABSC) is one of the most resistant bacteria against antimicrobial agents. The number of agents that can be used by oral route, such as macrolides, is limited in antimicrobial therapy. In recent years, rifabutin and clofazimine have gained importance as they can be administered by oral route and have shown synergistic effects with macrolides and aminoglycosides. The aim of this study was to determine the in vitro activity of rifabutin and clofazimine against clinical isolates of MABSC resistant to macrolides. A total of 48 MABSC isolates obtained from respiratory tract and other clinical samples in the Tuberculosis Laboratories of the Faculty of Medicine of Manisa Celal Bayar and Ege Universities were included in the study. Subspecies differentiation and aminoglycoside and macrolide resistance of the isolates were determined by GenoType NTM-DR test. Rifabutin and clofazimine susceptibilities were determined by standard broth microdilution method. Of the MABSC isolates 42 were identified as M.abscessus subsp. abscessus, three as M.abscessus subsp. bolletii and three as M.abscessus subsp. massiliense. None of the isolates exhibited rrs and rrl mutations indicating acquired macrolide resistance and aminoglycoside resistance. However, the erm(41) T28 genotype which is associated with inducible macrolide resistance was detected in 41 (85%) of the strains. All M.abscessus subsp. massiliense isolates were found to be genotypically susceptible to macrolides. The minimum inhibitory concentration (MIC) range values for rifabutin were 0.0625 to 32 µg/mL, while for clofazimine, the range was 0.0625 to 1 µg/mL. Rifabutin MIC values were significantly higher (mean 5.98 µg/mL vs 0.5 µg/mL, p= 0.026) in the isolates with macrolide resistance. There was no correlation between macrolide resistance and clofazimine MIC values (mean 0.25 µg/mL vs. 0.214 µg/mL, p= 0.758). The MIC50 and MIC90 values for rifabutin were 1 and 8 µg/mL, respectively, while for clofazimine they were 0.25 and 0.5 µg/mL. Macrolide resistance was found to be higher in isolates with rifabutin MIC values above the MIC50 value (p= 0.045). In conclusion, the determination of higher rifabutin MIC values in isolates resistant to macrolides suggested that susceptibility testing should be performed before adding rifabutin to the treatment regimen. The low MIC values of clofazimine in all strains indicated that it may be used as a first choice in the combination therapy. However, further studies using a larger number of clinical isolates and applying genotypic and phenotypic susceptibility tests are needed to determine threshold MIC values to assist clinicians in making treatment decisions.

The Inappropriateness of Using Rifampicin E-Test to Predict Rifabutin Resistance in Helicobacter pylori.

BACKGROUND: The aim of this study was to evaluate the rifamycin cross-resistance in Helicobacter pylori, and whether the use of rifampicin E-test strips to screen H. pylori rifabutin resistance is appropriate. METHODS: A total of 89 H. pylori isolates were included. Rifampicin minimum inhibitory concentrations (MICs) were obtained by E-test, while the MICs for rifapentine, rifaximin, and rifabutin were determined by agar dilution method. The rifamycin resistance rates based on different breakpoints were compared. Isolates with high-level rifampicin resistance were subjected to whole-genome sequencing. RESULTS: A wide distribution of MICs (mostly in the range 0.125-8 mg/L) was observed for rifampicin, rifapentine, and rifaximin. Using MIC >1, ≥ 4, and > 4 mg/L as the breakpoints, resistance rates to rifampicin/rifapentine/rifaximin were 60.4%/48.3%/38.2%, 28.1%/25.8%/23.6%, and 15.7%/16.9%/7.9%, respectively. However, the rifabutin MICs of all the tested H. pylori isolates were extremely low (≤0.016 mg/L). Applying MIC ≥ 0.125 mg/L as the breakpoint, rifabutin resistance was nil. No mutation was found in the rpoB gene sequences of the 2 isolates with high-level rifampicin resistance. CONCLUSIONS: There is a lack of cross-resistance between rifabutin and other rifamycins in H. pylori. The use of rifampicin E-test to predict H. pylori rifabutin resistance is inappropriate.

Structural diversity and biological relevance of benzenoid and atypical ansamycins and their congeners.

Covering: 2011 to 2021The structural division of ansamycins, including those of atypical cores and different lengths of the ansa chains, is presented. Recently discovered benzenoid and atypical ansamycin scaffolds are presented in relation to their natural source and biosynthetic routes realized in bacteria as well as their muta and semisynthetic modifications influencing biological properties. To better understand the structure-activity relationships among benzenoid ansamycins structural aspects together with mechanisms of action regarding different targets in cells, are discussed. The most promising directions for structural optimizations of benzenoid ansamycins, characterized by predominant anticancer properties, were discussed in view of their potential medical and pharmaceutical applications. The bibliography of the review covers mainly years from 2011 to 2021.

Rifabutin versus rifampicin bactericidal and antibiofilm activities against clinical strains of Staphylococcus spp. isolated from bone and joint infections.

BACKGROUND: Staphylococci account for approximately 60% of periprosthetic joint infections (PJIs). Rifampicin (RMP) combination therapy is generally considered to be the treatment of choice for staphylococcal PJIs but carries an important risk of adverse events and drug-drug interactions. Rifabutin (RFB) shares many of the properties of rifampicin but causes fewer adverse events. OBJECTIVES: To compare the minimal inhibitory concentration (MIC), the minimum bactericidal concentrations (MBC), and the minimum biofilm eradication concentrations (MBEC) of rifabutin and rifampicin for staphylococcal clinical strains isolated from PJIs. METHODS: 132 clinical strains of rifampicin-susceptible staphylococci [51 Staphylococcus aureus (SA), 48 Staphylococcus epidermidis (SE) and 33 other coagulase-negative staphylococci (CoNS)] were studied. The MBC and the MBEC were determined using the MBEC® Assay for rifabutin and rifampicin and were compared. RESULTS: When compared with the rifampicin MIC median value, the rifabutin MIC median value was significantly higher for SA (P < 0.05), but there was no statistically significant difference for SE (P = 0.25) and CoNS (P = 0.29). The rifabutin MBC median value was significantly higher than that of rifampicin for SA (P = 0.003) and was lower for SE (P = 0.003) and CoNS (P = 0.03). Rifabutin MBEC median value was statistically lower than that of rifampicin for all strains tested. CONCLUSIONS: Using the determination of MBEC values, our study suggests that rifabutin is more effective than rifampicin against clinical strains of Staphylococcus spp. obtained from PJIs. Using MBECs instead of MICs seems to be of interest when considering biofilms. In vivo higher efficacy of rifabutin when compared with rifampicin needs to be confirmed.

In vitro assessment of 17 antimicrobial agents against clinical Mycobacterium avium complex isolates.

BACKGROUND: Recently, Mycobacterium avium complex (MAC) infections have been increasing, especially in immunocompromised and older adults. The rapid increase has triggered a global health concern due to limited therapeutic strategies and adverse effects caused by long-term medication. To provide more evidence for the treatment of MAC, we studied the in vitro inhibitory activities of 17 antimicrobial agents against clinical MAC isolates. RESULTS: A total of 111 clinical MAC isolates were enrolled in the study and they were identified as M. intracellulare, M. avium, M. marseillense, M. colombiense, M. yongonense, and two isolates could not be identified at the species level. MAC strains had relatively low (0-21.6%) resistance to clarithromycin, amikacin, bedaquiline, rifabutin, streptomycin, and clofazimine, and the resistant rates to isoniazid, rifampin, linezolid, doxycycline, and ethionamide were very high (72.1-100%). In addition, M. avium had a significantly higher resistance rate than that of M. intracellulare for ethambutol (92.3% vs 40.7%, P < 0.001), amikacin (15.4% vs 1.2%, P = 0.049), and cycloserine (69.2% vs 25.9%, P = 0.004). CONCLUSIONS: Our results supported the current usage of macrolides, rifabutin, and aminoglycosides in the regimens for MAC infection, and also demonstrated the low resistance rate against new drugs, such as clofazimine, tedizolid, and bedaquiline, suggesting the possible implementation of these drugs in MAC treatment.

In vitro activity of rifabutin against Mycobacterium abscessus, clinical isolates.

The antibiotic options available for Mycobacterium abscessus (M. abscessus) infection are limited and no definitive therapeutic strategies have been formulated. The recent discovery that rifabutin is active against M. abscessus has raised interest in using rifabutin to treat this intractable disease. In this study, we evaluated the in vitro activity of rifabutin against 194 M. abscessus clinical isolates collected during 2012 January to 2017 December. As expected, rifabutin demonstrated considerably lower MICs against M. abscessus, with an MIC50 of 2 µg/mL and MIC90 of 4 µg/mL, respectively. Notably, the anti-M.abscessus activity was even stronger among clarithromycin-insusceptible strains. In addition, M. abscessus isolates with a rough morphotype were more sensitive to rifabutin compared with those forming smooth colonies when considered as a whole or in separate subspecies. Results from synergistic experiments revealed that the in vitro activity of rifabutin was significantly enhanced by the addition of amikacin, suggesting a promising strategy for M. abscessus infection combination treatment. Finally, five and three mutation patterns in rpoB and arr, respectively, were identified among the 194 strains through whole genome sequencing. However, none of them conferred rifabutin resistance. Our study is among the first to report the susceptibility of M. abscessus to rifabutin in vitro with a large amount of clinical isolates, suggesting that rifabutin is active, both alone and in combination, against M. abscessus and is worth considering as part of a combination treatment regimen for M. abscessus infections.

Low-Dose Rifabutin Increases Cytotoxicity in Antimitotic-Drug-Treated Resistant Cancer Cells by Exhibiting Strong P-gp-Inhibitory Activity.

The cytotoxicity of various antibiotics at low doses in drug-resistant cancer cells was evaluated. Low doses of rifabutin were found to markedly increase the cytotoxicity of various antimitotic drugs, such as vincristine (VIC), to P-glycoprotein (P-gp)-overexpressing antimitotic-drug-resistant KBV20C cells. Rifabutin was also found to exert high levels of P-gp-inhibitory activity at 4 and 24 h posttreatment, suggesting that the cytotoxicity of VIC + rifabutin was mainly due to the direct binding of rifabutin to P-gp and the reduction of VIC efflux by P-gp. The combination of VIC + rifabutin also increased early apoptosis, G2 arrest, and the DNA damaging marker, pH2AX protein. Interestingly, only the combination of VIC + rifabutin induced remarkable levels of cytotoxicity in resistant KBV20C cells, whereas other combinations (VIC + rifampin, VIC + rifapentine, and VIC + rifaximin) induced less cytotoxicity. Such finding suggests that rifabutin specifically increases the cytotoxicity of VIC in KBV20C cells, independent of the toxic effect of the ansamycin antibiotic. Only rifabutin had high P-gp-inhibitory activity, which suggests that its high P-gp-inhibitory activity led to the increased cytotoxicity of VIC + rifabutin. As rifabutin has long been used in the clinic, repositioning this drug for P-gp-overexpressing resistant cancer could increase the availability of treatments for patients with drug-resistant cancer.

Model-Based Comparative Analysis of Rifampicin and Rifabutin Drug-Drug Interaction Profile.

Rifamycins are widely used for treating mycobacterial and staphylococcal infections. Drug-drug interactions (DDI) caused by rifampicin (RIF) are a major issue. We used a model-based approach to predict the magnitude of DDI with RIF and rifabutin (RBT) for 217 cytochrome P450 (CYP) substrates. On average, DDI caused by low-dose RIF were twice as potent as those caused by RBT. Contrary to RIF, RBT appears unlikely to cause severe DDI, even with sensitive CYP substrates.

Blocking Bacterial Naphthohydroquinone Oxidation and ADP-Ribosylation Improves Activity of Rifamycins against Mycobacterium abscessus.

Rifampicin is an effective drug for treating tuberculosis (TB) but is not used to treat Mycobacterium abscessus infections due to poor in vitro activity. While rifabutin, another rifamycin, has better anti-M. abscessus activity, its activity is far from the nanomolar potencies of rifamycins against Mycobacterium tuberculosis. Here, we asked (i) why is rifabutin more active against M. abscessus than rifampicin, and (ii) why is rifabutin's anti-M. abscessus activity poorer than its anti-TB activity? Comparative analysis of naphthoquinone- versus naphthohydroquinone-containing rifamycins suggested that the improved activity of rifabutin over rifampicin is linked to its less readily oxidizable naphthoquinone core. Although rifabutin is resistant to bacterial oxidation, metabolite and genetic analyses showed that this rifamycin is metabolized by the ADP-ribosyltransferase ArrMab like rifampicin, preventing it from achieving the nanomolar activity that it displays against M. tuberculosis. Based on the identified dual mechanism of intrinsic rifamycin resistance, we hypothesized that rifamycins more potent than rifabutin should contain the molecule's naphthoquinone core plus a modification that blocks ADP-ribosylation at its C-23. To test these predictions, we performed a blinded screen of a diverse collection of 189 rifamycins and identified two molecules more potent than rifabutin. As predicted, these compounds contained both a more oxidatively resistant naphthoquinone core and C-25 modifications that blocked ADP-ribosylation. Together, this work revealed dual bacterial metabolism as the mechanism of intrinsic resistance of M. abscessus to rifamycins and provides proof of concept for the repositioning of rifamycins for M. abscessus disease by developing derivatives that resist both bacterial oxidation and ADP-ribosylation.

Rifabutin Is Inactivated by Mycobacterium abscessus Arr.

Mycobacterium abscessus exhibits Arr (ADP-ribosyltransferase)-dependent rifampin resistance. In apparent contrast, rifabutin (RBT) has demonstrated promising activity in M. abscessus infection models, implying that RBT might not be inactivated by Arr. RBT susceptibility testing of M. abscessusΔarr revealed a strongly decreased MIC. Our findings suggest that the efficacy of RBT might be enhanced by rendering RBT resilient to Arr-dependent modification or by blocking M. abscessus Arr activity.

Differential In Vitro Activities of Individual Drugs and Bedaquiline-Rifabutin Combinations against Actively Multiplying and Nutrient-Starved Mycobacterium abscessus.

Current treatment options for lung disease caused by Mycobacterium abscessus complex infections have limited effectiveness. To maximize the use of existing antibacterials and to help inform regimen design for treatment, we assessed the in vitro bactericidal activity of single drugs against actively multiplying and net nonreplicating M. abscessus populations in nutrient-rich and nutrient-starvation conditions, respectively. As single drugs, bedaquiline and rifabutin exerted bactericidal activity only against nutrient-starved and actively growing M. abscessus, respectively. However, when combined, both bedaquiline and rifabutin were able to specifically contribute bactericidal activity at relatively low, clinically relevant concentrations against both replicating and nonreplicating bacterial populations. The addition of a third drug, amikacin, further enhanced the bactericidal activity of the bedaquiline-rifabutin combination against nutrient-starved M. abscessus Overall, these in vitro data suggest that bedaquiline-rifabutin may be a potent backbone combination to support novel treatment regimens for M. abscessus infections. This rich data set of differential time- and concentration-dependent activity of drugs, alone and together, against M. abscessus also highlights several issues affecting interpretation and translation of in vitro findings.

Rifampin, Rifapentine, and Rifabutin Are Active against Intracellular Periprosthetic Joint Infection-Associated Staphylococcus epidermidis.

Staphylococcus epidermidis is a major cause of periprosthetic joint infection (PJI); its intracellular persistence within osteoblasts may compromise therapy if that therapy is not intracellularly active. The intracellular activity of rifampin, rifapentine, and rifabutin was assessed against five rifampin-susceptible and two rifampin-resistant S. epidermidis isolates. Compared to no treatment, treatment resulted in a ≥2-fold log10 reduction of intracellular rifampin-susceptible, but not rifampin-resistant, S. epidermidis These findings show activity of rifampin, rifapentine, and rifabutin against intraosteoblast PJI-associated S. epidermidis.

Synergistic Rifabutin and Colistin Reduce Emergence of Resistance When Treating Acinetobacter baumannii.

Recently, we reported rifabutin hyperactivity against Acinetobacter baumannii We sought to characterize potential interactions between rifabutin and colistin, the last-resort drug for carbapenem-resistant infections. Rifabutin and colistin were synergistic in vitro and in vivo, and low-dose colistin significantly suppressed emergence of resistance to rifabutin. Thus, this combination is a promising therapeutic option for highly resistant A. baumannii infections.