RESUMEN
Spectinomycin is an aminocyclitol antibiotic with a unique ribosomal binding site. Prior synthetic modifications of spectinomycin have enhanced potency and antibacterial spectrum through addition at the 6'-position to produce trospectomycin and to the 3'-position to produce spectinamides and aminomethyl spectinomycins. This study focused on the design, synthesis, and evaluation of three 3',6'-disubstituted spectinomycin analogs: trospectinamide, N-benzyl linked aminomethyl, and N-ethylene linked aminomethyl trospectomycins. Computational experiments predicted that these disubstituted analogs would be capable of binding within the SPC ribosomal binding site. The new analogs were synthesized from trospectomycin, adapting the previously established routes for the spectinamide and aminomethyl spectinomycin series. In a cell-free translation assay, the disubstituted analogs showed ribosomal inhibition similar to spectinomycin or trospectomycin. These disubstituted analogs demonstrated inhibitory MIC activity against various bacterial species with the 3'-modification dictating spectrum of activity, leading to improved activity against mycobacterium species. Notably, N-ethylene linked aminomethyl trospectomycins exhibited increased potency against Mycobacterium abscessus and trospectinamide displayed robust activity against M. tuberculosis, aligning with the selective efficacy of spectinamides. The study also found that trospectomycin is susceptible to efflux in M. tuberculosis and M. abscessus. These findings contribute to the understanding of the structure-activity relationship of spectinomycin analogs and can guide the design and synthesis of more effective spectinomycin compounds.
Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Espectinomicina , Espectinomicina/farmacología , Espectinomicina/análogos & derivados , Espectinomicina/síntesis química , Antibacterianos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Relación Estructura-Actividad , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Sitios de Unión , Bacterias/efectos de los fármacosRESUMEN
Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.
Asunto(s)
Antiinfecciosos , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Humanos , Animales , Ratones , Espectinomicina/farmacología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Antibacterianos/farmacología , Micobacterias no Tuberculosas , Antiinfecciosos/farmacología , Etilenos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Fidaxomicin (Fdx) is a natural product antibiotic with potent activity against Clostridioides difficile and other Gram-positive bacteria such as Mycobacterium tuberculosis. Only a few Fdx derivatives have been synthesized and examined for their biological activity in the 50â years since its discovery. Fdx has a well-studied mechanism of action, namely inhibition of the bacterial RNA polymerase. Yet, the targeted organisms harbor different target protein sequences, which poses a challenge for the rational development of new semisynthetic Fdx derivatives. We introduced substituents on the two phenolic hydroxy groups of Fdx and evaluated the resulting trends in antibiotic activity against M.â tuberculosis, C.â difficile, and the Gram-negative model organism Caulobacter crescentus. As suggested by the target protein structures, we identified the preferable derivatisation site for each organism. The derivative ortho-methyl Fdx also exhibited activity against the Gram-negative C.â crescentus wild type, a first for fidaxomicin antibiotics. These insights will guide the synthesis of next-generation fidaxomicin antibiotics.
Asunto(s)
Clostridioides difficile , Mycobacterium tuberculosis , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Fidaxomicina , Aminoglicósidos/farmacología , ARN Polimerasas Dirigidas por ADNRESUMEN
Recent outbreaks of cardiac surgery-associated Mycobacterium chimaera infections have highlighted the importance of species differentiation within the Mycobacterium avium complex and pointed to a lack of antibiotic susceptibility data for M. chimaera Using the MGIT 960/EpiCenter TB eXiST platform, we have determined antibiotic susceptibility patterns of 48 clinical M. chimaera isolates and 139 other non-tuberculous mycobacteria including 119 members of the M. avium complex and 20 Mycobacterium kansasii towards clofazimine and other drugs used to treat infections with slowly growing nontuberculous mycobacteria (NTM). MIC50, MIC90 and tentative epidemiological cutoff (ECOFF) values for clofazimine were 0.5 mg/L, 1 mg/L and 2 mg/L for M. chimaera. Comparable values were observed for other M. avium complex members, lower MIC50 (≤0.25 mg/L), MIC90 (0.5 mg/L) and ECOFF (1 mg/L) values were found for M. kansasii Susceptibility to clarithromycin, ethambutol, rifampin, rifabutin, amikacin, moxifloxacin and linezolid was in general similar for M. chimaera and other members of the M. avium complex but increased for M. kansasii The herein determined MIC distributions, MIC90 and ECOFF values of clofazimine for M. chimaera and other NTM provide the basis for the definition of clinical breakpoints. Further studies are needed to establish correlation of in vitro susceptibility and clinical outcome.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Humanos , Aminoglicósidos/farmacología , Aminoglicósidos/uso terapéutico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Microbiana , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Pruebas de Sensibilidad Microbiana , Farmacorresistencia BacterianaRESUMEN
Bedaquiline and clofazimine are increasingly used to treat infections with Mycobacterium abscessus. We determined distributions of MICs by broth microdilution for bedaquiline and clofazimine for 61 M. abscessus clinical isolates using different media and incubation times. We show that incubation time and growth media critically influence the MIC. Our data will aid in defining future clinical breakpoints for in vitro susceptibility testing for bedaquiline and clofazimine in M. abscessus.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Clofazimina/farmacología , Clofazimina/uso terapéutico , Diarilquinolinas/farmacología , Diarilquinolinas/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiologíaRESUMEN
Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1ß in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.
Asunto(s)
Vacuna BCG/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Pulmón/inmunología , Animales , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Vacuna BCG/administración & dosificación , Reactividad Cruzada , Femenino , Inflamación/inmunología , Inyecciones Intradérmicas , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mycobacterium bovis/inmunología , Fármacos Fotosensibilizantes/administración & dosificación , Factor de Necrosis Tumoral alfa/biosíntesis , Vacunación/métodosRESUMEN
Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's inherent resistance to clinically available antimicrobials. The low bactericidal potency of currently available treatment regimens is of concern and testifies to the poor therapeutic outcomes for pulmonary M. abscessus infections. Mechanistically, we demonstrate here that the acetyltransferase Eis2 is responsible for the lack of bactericidal activity of amikacin, the standard aminoglycoside used in combination treatment. In contrast, the aminoglycoside apramycin, with a distinct structure, is not modified by any of the pathogen's innate aminoglycoside resistance mechanisms and is not affected by the multidrug resistance regulator WhiB7. As a consequence, apramycin uniquely shows potent bactericidal activity against M. abscessus. This favorable feature of apramycin is reflected in a mouse model of pulmonary M. abscessus infection, which demonstrates superior activity, compared with amikacin. These findings encourage the development of apramycin for the treatment of M. abscessus infections and suggest that M. abscessus eradication in pulmonary disease may be within therapeutic reach.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Nebramicina , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Ratones , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Nebramicina/análogos & derivados , Nebramicina/farmacología , Nebramicina/uso terapéuticoRESUMEN
Genome mining and bioactivity studies suggested the sponge-derived bacterium Aquimarina sp. Aq135 as a producer of new antibiotics. Activity-guided isolation identified antibacterial peptides, named aquimarins, featuring a new scaffold with an unusual C-terminal amino group and chlorine moieties. Responsible for the halogenation is the FeII /α-ketoglutarate-dependent chlorinase AqmA that halogenates up to two isoleucine residues in a carrier protein-dependent fashion. Total syntheses of two natural aquimarins and eight non-natural variants were developed. Structure-activity relationship (SAR) studies with these compounds showed that the synthetically more laborious chlorinations are not required for antibacterial activity but enhance cytotoxicity. In contrast, variants lacking the C-terminal amine were virtually inactive, suggesting diamines similar to the terminal aquimarin residue as candidate building blocks for new peptidomimetic antibiotics.
Asunto(s)
Antibacterianos/química , Flavobacteriaceae/química , Péptidos/química , Antibacterianos/metabolismo , Conformación Molecular , Péptidos/genética , Péptidos/metabolismo , EstereoisomerismoRESUMEN
Mycobacterium tuberculosis (Mtb) inhibits autophagy to promote its survival in host cells. However, the molecular mechanisms by which Mtb inhibits autophagy are poorly understood. Here, we report a previously unknown mechanism in which Mtb phosphoribosyltransferase (MtbPRT) inhibits autophagy in an mTOR, negative regulator of autophagy, independent manner by inducing histone hypermethylation (H3K9me2/3) at the Atg5 and Atg7 promoters by activating p38-MAPK- and EHMT2 methyltransferase-dependent signaling pathways. Additionally, we find that MtbPRT induces EZH2 methyltransferase-dependent H3K27me3 hypermethylation and reduces histone acetylation modifications (H3K9ac and H3K27ac) by upregulating histone deacetylase 3 to inhibit autophagy. In summary, this is the first demonstration that Mtb inhibits autophagy by inducing histone hypermethylation in autophagy-related genes to promote intracellular bacterial survival.
Asunto(s)
Histonas , Macrófagos/microbiología , Mycobacterium tuberculosis , Pentosiltransferasa/metabolismo , Autofagia , Histonas/metabolismo , Macrófagos/metabolismo , Metilación , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Transducción de SeñalRESUMEN
Fidaxomicin (FDX) is a marketed antibiotic for the treatment of Clostridioides difficile infections (CDI). Fidaxomicin displays antibacterial properties against many Gram-positive bacteria, yet the application of this antibiotic is currently limited to treatment of CDI. Semisynthetic modifications present a promising strategy to improve its pharmacokinetic properties and also circumvent resistance development by broadening the structural diversity of the derivatives. Here, based on a rational design using cryo-EM structural analysis, we implement two strategic site-selective catalytic reactions with a special emphasis to study the role of the carbohydrate units. Site-selective introduction of various ester moieties on the noviose as well as a Tsuji-Trost type rhamnose cleavage allow the synthesis of novel fidaxomicin analogs with promising antibacterial activities against C. difficile and Mycobacterium tuberculosis.
RESUMEN
BACKGROUND: Drug resistance threatens global tuberculosis control. We aimed to examine mortality in patients with tuberculosis from high-burden countries, according to concordance or discordance of results from drug susceptibility testing done locally and whole-genome sequencing (WGS). METHODS: In this multicentre cohort study, we collected pulmonary Mycobacterium tuberculosis isolates and clinical data from individuals with tuberculosis from antiretroviral therapy programmes and tuberculosis clinics in Côte d'Ivoire, Democratic Republic of the Congo, Kenya, Nigeria, Peru, South Africa, and Thailand, stratified by HIV status and drug resistance. Sites tested drug susceptibility using routinely available methods. WGS was done on Illumina HiSeq 2500 in the USA and Switzerland, and TBprofiler was used to analyse the genomes. We included individuals aged 16 years or older with pulmonary tuberculosis (bacteriologically confirmed or clinically diagnosed). We analysed mortality in multivariable logistic regression models adjusted for sex, age, HIV status, history of tuberculosis, and sputum positivity. FINDINGS: Between Sept 1, 2014, and July 4, 2016, of 634 patients included in our previous analysis, we included 582 patients with tuberculosis (median age 33 years [IQR 27-43], 225 [39%] women, and 247 [42%] HIV-positive). Based on WGS, 339 (58%) isolates were pan-susceptible, 35 (6%) monoresistant, 146 (25%) multidrug-resistant, and 24 (4%) pre-extensively drug-resistant (pre-XDR) or XDR. The analysis of mortality was based on 530 patients; 63 (12%) died and 77 (15%) patients received inappropriate treatment. Mortality ranged from 6% (18 of 310) in patients with pan-susceptible tuberculosis to 39% (nine of 23) in patients with pre-XDR or XDR tuberculosis. The adjusted odds ratio for mortality was 4·92 (95% CI 2·47-9·78) among undertreated patients, compared with appropriately treated patients. INTERPRETATION: In seven countries with a high burden of tuberculosis, we observed discrepancies between drug resistance patterns obtained locally and WGS. The underdiagnosis of drug resistance resulted in inappropriate treatment and higher mortality. WGS can provide accurate and detailed drug resistance information required to improve the outcomes of drug-resistant tuberculosis in high-burden settings. Our results support WHO's call for point-of-care tests based on WGS. FUNDING: National Institutes of Allergy and Infectious Diseases, Swiss National Science Foundation, and Swiss National Center for Mycobacteria.
Asunto(s)
Infecciones por VIH , Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis Pulmonar , Tuberculosis , Adulto , Antituberculosos/farmacología , Estudios de Cohortes , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Pulmonar/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , ADP Ribosa Transferasas , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Mycobacterium abscessus/genética , Rifabutina/farmacología , Rifampin/farmacologíaRESUMEN
The glycoslated macrocyclic antibiotic fidaxomicin (1, tiacumicin B, lipiarmycin A3) displays good to excellent activity against Gram-positive bacteria and was approved for the treatment of Clostridium difficile infections (CDI). Among the main limitations for this compound, its low water solubility impacts further clinical uses. We report on the synthesis of new fidaxomicin derivatives based on structural design and utilizing an operationally simple one-step protecting group-free preparative approach from the natural product. An increase in solubility of up to 25-fold with largely retained activity was observed. Furthermore, hybrid antibiotics were prepared that show improved antibiotic activities.
RESUMEN
Mutations in the genes of the F420 signaling pathway of Mycobacterium tuberculosis complex, including dnn, fgd1, fbiA, fbiB, fbiC, and fbiD, can lead to delamanid resistance. We searched for such mutations among 129 M. tuberculosis strains from Asia, South America, and Africa using whole-genome sequencing; 70 (54%) strains had at least one mutation in one of the genes. For 10 strains with mutations, we determined the MIC of delamanid. We found one strain from a delamanid-naive patient carrying the natural polymorphism Tyr29del (ddn) that was associated with a critical delamanid MIC.
Asunto(s)
Mycobacterium tuberculosis , Preparaciones Farmacéuticas , Tuberculosis Resistente a Múltiples Medicamentos , África , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Asia , Humanos , Pruebas de Sensibilidad Microbiana , Mutación/genética , Mycobacterium tuberculosis/genética , Nitroimidazoles , Oxazoles , América del Sur , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
Drug resistance in Mycobacterium tuberculosis is relentlessly progressing while only a handful of novel drug candidates are developed. Here we describe a GFP-based high-throughput screening of 386,496 diverse compounds to identify putative tuberculosis drug candidates. In an exploratory analysis of the model organism M. bovis BCG and M. smegmatis and the subsequent screening of the main library, we identified 6354 compounds with anti-mycobacterial activity. These hit compounds were predominantly selective for mycobacteria while dozens had activity in the low µM range. We tested toxicity against the human monocyte/macrophage cell line THP-1 and elaborated activity against M. tuberculosis growing in liquid broth, under unique conditions such as non-replicating persistence or inhibition of M. tuberculosis residing in macrophages. Finally, spontaneous compound-resistant M. tuberculosis mutants were selected and subsequently analyzed by whole genome sequencing. In addition to compounds targeting the well-described proteins Pks13 and MmpL3, we identified two novel scaffolds targeting the bifunctional guanosine pentaphosphate synthetase/ polyribonucleotide nucleotidyltransferase GpsI, or interacting with the aminopeptidase PepB, a probable pro-drug activator. KEY MESSAGES: A newly identified scaffold targets the bifunctional enzyme GpsI. The aminopeptidase PepB is interacting with a second novel scaffold. Phenotypic screenings regularly identify novel compounds targeting Pks13 and MmpL3.
Asunto(s)
Mycobacterium bovis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Antibacterianos/farmacología , Humanos , Kanamicina/metabolismo , Ligasas/metabolismo , Macrófagos/metabolismo , Pruebas de Sensibilidad Microbiana , Células THP-1 , Secuenciación Completa del GenomaRESUMEN
The gastric lamina propria of mice that have been experimentally infected with the pathobiont Helicobacter pylori hosts a dense network of myeloid cells that includes BATF3-dependent CD103+ dendritic cells (DCs). We show here that CD103+ DCs are strictly required for gastric Th1 responses to H. pylori and for H. pylori infection control. A similar dependence of type 1 immunity on CD103+ DCs is observed in a Mycobacterium bovis BCG infection model, and in a syngeneic colon cancer model. Strikingly, we find that not only the expansion and/or recruitment of Th1 cells, but also of peripherally induced, neuropilin-negative regulatory T-cells to sites of infection requires BATF3-dependent DCs. A shared feature of the examined models is the strongly reduced production of the chemokines and CXCR3 ligands CXCL9, 10 and 11 in BATF3-deficient mice. The results implicate BATF3-dependent DCs in the recruitment of CXCR3+ effector and regulatory T-cells to target tissues and in their local expansion.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Células Dendríticas/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Mycobacterium bovis/inmunología , Proteínas Represoras/inmunología , Linfocitos T Reguladores/inmunología , Tuberculosis/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular Tumoral , Quimiocinas CXC/genética , Quimiocinas CXC/inmunología , Células Dendríticas/microbiología , Células Dendríticas/patología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/patología , Ratones , Ratones Noqueados , Receptores CXCR3/genética , Receptores CXCR3/inmunología , Proteínas Represoras/genética , Linfocitos T Reguladores/microbiología , Linfocitos T Reguladores/patología , Tuberculosis/genética , Tuberculosis/patologíaRESUMEN
Whole-genome sequencing allows rapid detection of drug-resistant Mycobacterium tuberculosis isolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinical M. tuberculosis isolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/genética , Antituberculosos/farmacología , República Democrática del Congo , Etambutol/farmacología , Genoma Bacteriano/genética , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación/genética , Mycobacterium tuberculosis/efectos de los fármacos , Perú , Fenotipo , Suiza , Tailandia , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Secuenciación Completa del Genoma/métodosRESUMEN
The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two-gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon's function. Interestingly, influx of the fluorescent dyes BCECF-AM and calcein-AM in de-energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.