RESUMEN
For the past decades, an acidic pH has been used to render Mycobacterium tuberculosis susceptible to pyrazinamide for in vitro testing. Here, we show that at the standard breakpoint concentration and reduced culture temperatures, pyrazinamide (PZA) is active against tuberculosis (TB) at neutral pH. This finding should help unravel the mechanism of action of PZA and allow drug susceptibility testing (DST) methods to be optimized.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Pirazinamida/farmacología , Tuberculosis/tratamiento farmacológico , Humanos , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana/métodos , TemperaturaRESUMEN
BACKGROUND: Many patients treated for tuberculosis (TB) in low and middle income countries are treated based on clinical suspicion without bacteriological confirmation. This is often due to lack of rapid simple accurate diagnostics and low healthcare provider confidence in the predictive value of current tests. We previously reported in an animal TB model that levels of host markers rapidly change in response to treatment initiation. METHODS: We assessed the potential of host biomarker kinetics of TB patients during the first two weeks of therapy to identify patients responding to treatment. Adult patients clinically diagnosed with and treated for TB, 29 in Nigeria and 24 in Nepal, were analyzed. RESULTS: Changes in concentrations of non-specific host biomarkers, particularly IP-10, in response to the first week of anti-TB therapy were strongly associated with bacteriological confirmation of TB. A decrease in IP-10 level of >300pg/ml between 0 and 7 days of treatment identified 75% of both smear-positive and smear-negative culture positive patients and correctly excluded TB in all nine culture negative patients. CONCLUSIONS: Monitoring of early IP-10 responses to treatment could form the basis of a simplified assay and could help identify patients who were erroneously clinically diagnosed with TB or those infected with drug resistant strains on inappropriate treatment. We believe this approach may be particularly appropriate for difficult to diagnose patients, e.g. smear-negative HIV-positive, or those with extra-pulmonary TB, often treated without bacterial confirmation.
Asunto(s)
Antituberculosos/uso terapéutico , Citocinas/sangre , Tuberculosis/sangre , Tuberculosis/tratamiento farmacológico , Adolescente , Adulto , Anciano , Autoanticuerpos/sangre , Biomarcadores , Quimiocina CXCL10/sangre , Coinfección , Femenino , Estudios de Seguimiento , Seropositividad para VIH , Humanos , Cinética , Masculino , Persona de Mediana Edad , Pronóstico , Curva ROC , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Tuberculosis/diagnóstico , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/tratamiento farmacológico , Adulto JovenRESUMEN
In contrast to most other antimycobacterial drugs where--particularly in multidrug-resistant (MDR) strains--a limited number of resistance mutations dominate, pyrazinamide (PZA) resistance associated mutations remain highly diverse with limited clustering. This apparent lack of evolutionary selection for successful PZA resistance mechanisms deserves attention. A clear understanding of the epidemiology of PZA resistance acquisition and spread would be expected to result in important insights into how PZA might be better exploited in treatment regimens to minimize the amplification of Mycobacterium tuberculosis (MTB) drug resistance. We propose that PZA resistance typically induces a fitness cost that impairs MTB transmission. This would explain the lack of extensive clustering for PZA-resistant mutants. Our hypothesis also leads to a series of testable predictions which we outline that could confirm or refute our ideas.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana , Mycobacterium tuberculosis/efectos de los fármacos , Pirazinamida/farmacología , Transmisión de Enfermedad Infecciosa , Humanos , Mycobacterium tuberculosis/fisiología , Tuberculosis/microbiología , Tuberculosis/transmisión , VirulenciaRESUMEN
BACKGROUND: Due to the increasing prevalence of Mycobacterium tuberculosis strains resistant to one or more antibiotics, there is a need for new quantitative culture methods both for drug susceptibility testing and for validation of mutations putatively associated with drug resistance. We previously developed a (myco) bacterial culture method, in which multiple growing microcolonies are monitored individually. Transfer of the growing microcolonies to selective medium allows the effect on the growth rate of each individual colony to be determined. As entire growing colonies are exposed to antibiotics rather than re-subbed, a second lag phase is avoided and results are obtained more rapidly. Here we investigate the performance of the microcolony method to differentiate between ethambutol (EMB) resistant, intermediate and susceptible strains. METHODS: One week old microcolonies from a reference panel of four strains with known EMB susceptibility were transferred to different concentrations of EMB. Growth rates during the 1st 2 days of exposure were used to set up classification criteria to test and classify a blinded panel of 20 tuberculosis strains with different susceptibilities. RESULTS: For 18 strains (90%) reference culture results corresponded to our classifications based on data collected within 9 days of inoculation. A single strain was classified as Intermediate instead of Susceptible, and 1 strain could not be classified due to a contamination. CONCLUSIONS: Using a microcolony growth monitoring method we were able to classify, within 9 days after inoculation, a panel of strains as EMB susceptible, intermediate or resistant with 90% correlation to the reference methods.
Asunto(s)
Antituberculosos/farmacología , Etambutol/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Farmacorresistencia Bacteriana , HumanosRESUMEN
Recently we proposed exploring the potential of treatment stimulated testing as diagnostic method for tuberculosis (TB). An infection controlled placebo controlled mouse study was performed to investigate whether serum cytokine levels changed measurably during the early phase of TB chemotherapy. Serum was collected prior to and during the first 3 weeks of isoniazid (INH) and rifampicin (RIF) chemotherapy, and levels of 23 selected cytokines/chemokines were measured using a liquid bead array. The serum levels of IFNγ, IP-10, MIG, MCP-1, IL-17 and IL-6 were elevated in the TB infected mice compared to non-infected mice at least at 1 time point measured. In infected mice, IFNγ, IP-10, MIG and MCP-1 levels decreased within 7 days of treatment with RIF+INH compared to placebo. Treatment of non-infected mice in the absence of tuberculosis infection had no effect on these cytokines. IL-17 and IL-6 had decreased to baseline in all infected mice prior to the initiation of treatment. This study demonstrates that systemic levels of some cytokines, more specifically IFNγ, IP-10, MIG and MCP-1, rapidly and specifically change upon starting TB chemotherapy only in the presence of infection in a mouse model. Thus, IFNγ, IP-10, MIG and MCP-1 are promising 'Treat-to-Test' targets for the diagnosis of TB and deserve further investigation in a study on human TB suspects.
Asunto(s)
Especificidad del Huésped , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Antituberculosos/uso terapéutico , Citocinas/metabolismo , Femenino , Isoniazida/farmacología , Isoniazida/uso terapéutico , Metagenoma/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Placebos , Rifampin/farmacología , Rifampin/uso terapéutico , Tuberculosis Pulmonar/metabolismo , Tuberculosis Pulmonar/microbiologíaAsunto(s)
Antituberculosos/uso terapéutico , Biomarcadores/análisis , Tuberculosis/diagnóstico , Tuberculosis/tratamiento farmacológico , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/análisis , Humanos , Pruebas Inmunológicas , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Even with the advent of nucleic acid (NA) amplification technologies the culture of mycobacteria for diagnostic and other applications remains of critical importance. Notably microscopic observed drug susceptibility testing (MODS), as opposed to traditional culture on solid media or automated liquid culture, has shown potential to both speed up and increase the provision of mycobacterial culture in high burden settings. METHODS: Here we explore the growth of Mycobacterial tuberculosis microcolonies, imaged by automated digital microscopy, cultured on a porous aluminium oxide (PAO) supports. Repeated imaging during colony growth greatly simplifies "computer vision" and presumptive identification of microcolonies was achieved here using existing publically available algorithms. Our system thus allows the growth of individual microcolonies to be monitored and critically, also to change the media during the growth phase without disrupting the microcolonies. Transfer of identified microcolonies onto selective media allowed us, within 1-2 bacterial generations, to rapidly detect the drug susceptibility of individual microcolonies, eliminating the need for time consuming subculturing or the inoculation of multiple parallel cultures. SIGNIFICANCE: Monitoring the phenotype of individual microcolonies as they grow has immense potential for research, screening, and ultimately M. tuberculosis diagnostic applications. The method described is particularly appealing with respect to speed and automation.
Asunto(s)
Automatización , Mycobacterium tuberculosis/aislamiento & purificación , Medios de Cultivo , Mycobacterium tuberculosis/crecimiento & desarrolloRESUMEN
Cathelicidins are effector molecules of the innate host defense system that establish an antimicrobial barrier at epithelial interfaces. The human cathelicidin LL-37, in addition to its antimicrobial activity, also exhibits immunomodulatory effects, such as inhibition of pro-inflammatory responses to bacterial LPS in human monocytic cells. In this report, we demonstrate that LL-37 almost completely prevents the pro-inflammatory cytokine release by human peripheral blood mononuclear cells (PBMCs) following stimulation with Toll-like receptor (TLR)4 and TLR2/1 agonists while leaving TLR2/6, TLR5, TLR7 and TLR8 responses unchanged. Modulation of the TLR response by LL-37 occurred at least partly through the MAP kinase pathway via inhibition of p38 phosphorylation. By using an LL-37 library with overlapping sequences, we identified the mid-region of LL-37, comprising amino acids 13-31, as the active domain for the modulation of TLR responses. The mechanism of immunomodulation of LL-37 and LL-37 fragments is lipopoly-saccharide binding. Correlations between the capacity of LL-37 fragments to modulate TLR responses and their physico-chemical properties revealed that cationicity and hydrophobicity are essential for the modulation of LL-37-mediated TLR responses.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Receptores Toll-Like/química , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Catelicidinas , Células Cultivadas , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Humanos , Ligandos , Datos de Secuencia Molecular , Transducción de Señal , Relación Estructura-Actividad , Receptores Toll-Like/agonistas , Receptores Toll-Like/efectos de los fármacos , Receptores Toll-Like/metabolismoRESUMEN
The human cathelicidin peptide LL-37 and several truncated variants differ in their capability to transmigrate over the plasma membrane of Candida albicans. We investigated whether retention at the cell perimeter or membrane transmigration affects their membrane-disrupting activities and candidacidal properties. Using fluorescein-labeled peptides, we demonstrate that LL-37 and its C-terminally truncated peptide LL-31 remain permanently associated with the perimeter of the cell. The N-terminally truncated peptide RK-31 initially accumulated at the cell boundary, but transmigrated into the cytoplasm within 30 min. The C-terminally truncated peptide LL-25 transmigrated instantaneously into the cytoplasm. The ultrastructural effects on the plasma membrane were studied by freeze-fracture electron microscopy combined with filipin cytochemistry. All peptides, whether they transmigrated over the plasma membrane or not, induced phase separation in the plasma membrane. All peptides induced leakage of cell components, including nucleotides and proteins. Proteins were identified by SDS-PAGE in combination with mass spectrometry, which revealed that predominantly proteins smaller than 50 kDa had leaked out of C. albicans.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Candida albicans/metabolismo , Membrana Celular/metabolismo , Variación Genética/genética , Metabolismo de los Lípidos , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Candida albicans/ultraestructura , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular , Humanos , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Alineación de Secuencia , CatelicidinasRESUMEN
The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process.
Asunto(s)
Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Candida albicans/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Proteínas y Péptidos Salivales/farmacología , Histatinas , Nucleótidos , CatelicidinasRESUMEN
The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an indicator for ROS. With both peptides, a substantial enhancement of fluorescence was observed. However, TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a cell-permeant ROS scavenger, did not have an inhibitory effect on killing or on the enhancement of fluorescence. Furthermore, antimycin and azide, which have been reported to induce ROS in vitro, were not able to enhance the dihydroethidium fluorescence, while chlorhexidine, a non-specific antiseptic agent, enhanced dihydroethidium fluorescence to the same extent as did the peptides. Fluorescence microscopy showed the fluorescence enhancement to be a consequence of the release of unbound preformed ethidium from the mitochondrial matrix within the cell. It is concluded that ROS do not play a role in the histatin 5-mediated killing of C. albicans.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Etidio/análogos & derivados , Especies Reactivas de Oxígeno/metabolismo , Proteínas y Péptidos Salivales/farmacología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Óxidos N-Cíclicos/farmacología , Etidio/metabolismo , Colorantes Fluorescentes/metabolismo , Histatinas , Pruebas de Sensibilidad Microbiana/métodos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Proteínas y Péptidos Salivales/química , Proteínas y Péptidos Salivales/metabolismoRESUMEN
A number of cationic antimicrobial peptides, among which are histatin 5 and the derived peptides dhvar4 and dhvar5, enter their target cells and interact with internal organelles. There still are questions about the mechanisms by which antimicrobial peptides translocate across the membrane. We used a liposome model to study membrane binding, translocation and membrane-perturbing capacities of histatin 5, dhvar4 and dhvar5. Despite the differences in amphipathic characters of these peptides, they bound equally well to liposomes, whereas their membrane activities differed remarkably: dhvar4 translocated at the fastest rate, followed by dhvar5, whereas the histatin 5 translocation rate was much lower. The same pattern was seen for the extent of calcein release: highest with dhvar4, less with dhvar5 and almost none with histatin 5. The translocation and disruptive actions of dhvar5 did not seem to be coupled, because translocation occurred on a much longer timescale than calcein release, which ended within a few minutes. We conclude that peptide translocation can occur through peptide-phospholipid interactions, and that this is a possible mechanism by which antimicrobial peptides enter cells. However, the translocation rate was much lower in this model membrane system than that seen in yeast cells. Thus it is likely that, at least for some peptides, additional features promoting the translocation across biological membranes are involved as well.