RESUMEN
Mycobacterium smegmatis is intrinsically resistant to thiacetazone, an anti-tubercular thiourea; however we report here that it causes a mild inhibition in growth in liquid medium. Since mycolic acid biosynthesis was affected, we cloned and expressed Mycobacterium smegmatis mycolic acid methyltransferases, postulated as targets for thiacetazone in other mycobacterial species. During this analysis we identified MSMEG_1350 as the methyltransferase involved in epoxy mycolic acid synthesis since its deletion led to their total loss. Phenotypic characterization of the mutant strain showed colony morphology alterations at all temperatures, reduced growth and a slightly increased susceptibility to SDS, lipophilic and large hydrophilic drugs at 20 °C with little effect at 37 °C. No changes were detected between parental and mutant strains in biofilm formation, sliding motility or sedimentation rate. Intriguingly, we found that several mycobacteriophages severely decreased their ability to form plaques in the mutant strain. Taken together our results prove that, in spite of being a minor component of the mycolic acid pool, epoxy-mycolates are required for a proper assembly and functioning of the cell envelope. Further studies are warranted to decipher the role of epoxy-mycolates in the M. smegmatis cell envelope.
Asunto(s)
Proteínas Bacterianas/genética , Metiltransferasas/genética , Micobacteriófagos/fisiología , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/virología , Ácidos Micólicos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Frío , Metiltransferasas/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Mycobacterium smegmatis/fisiología , Eliminación de SecuenciaRESUMEN
Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc(2) 155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.
Asunto(s)
Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Micobacteriófagos/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/virología , Electroporación , Proteínas Fluorescentes Verdes/metabolismo , Lisogenia , Micobacteriófagos/fisiología , Mycobacterium smegmatis/metabolismo , Regiones Promotoras Genéticas , Eliminación de SecuenciaRESUMEN
Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100-400 nm, genome size in the 50-70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.
Asunto(s)
Micobacteriófagos/genética , Mycobacterium/virología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antituberculosos/farmacología , Cationes/metabolismo , Biología Computacional , ADN Viral , Orden Génico , Genoma Viral , Isoniazida/farmacología , Datos de Secuencia Molecular , Micobacteriófagos/clasificación , Micobacteriófagos/aislamiento & purificación , Micobacteriófagos/fisiología , Filogenia , Alineación de Secuencia , Ensayo de Placa Viral , Proteínas Virales/química , Proteínas Virales/genética , Tropismo Viral , Replicación Viral/efectos de los fármacosRESUMEN
We tested a mycobacteriophage D29-based method for fluoroquinolone susceptibility assessment in clinical isolates of Mycobacteriumtuberculosis. The method was incapable of identifying susceptible strains as such, although a slightly different published protocol successfully identified resistant and susceptible strains. Thus, caution is necessary when choosing an "in-house" D29-based protocol for testing of drug resistance.
Asunto(s)
Fluoroquinolonas/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Micobacteriófagos/fisiología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/virología , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Micobacteriófagos/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Resistance to commonly used antituberculosis drugs is emerging worldwide. Conventional drug-susceptibility testing (DST) methods are slow and demanding. Alternative, rapid DST methods would permit the early detection of drug resistance and, in turn, arrest tuberculosis transmission. METHODS: A cost-effectiveness analysis of 5 DST methods was performed in the context of a clinical trial that compared rapid with conventional DST methods. The methods under investigation were direct phage-replication assay (FASTPlaque-Response; Biotech), direct amplification and reverse hybridization of the rpoB gene (INNO-LiPA; Innogenetics), indirect colorimetric minimum inhibitory concentration assay (MTT; ICN Biomedicals), and direct proportion method on Löwenstein-Jensen medium. These were compared with the widely used indirect proportion method on Löwenstein-Jensen medium. RESULTS: All alternative DST methods were found to be cost-effective, compared with other health care interventions. DST methods also generate substantial cost savings in settings of high prevalence of multidrug-resistant tuberculosis. Excluding the effects of transmission, the direct proportion method on Löwenstein-Jensen medium was the most cost-effective alternative DST method for patient groups with prevalences of multidrug-resistant tuberculosis of 2%, 5%, 20%, and 50% (cost in US$2004, $94, $36, $8, and $2 per disability-adjusted life year, respectively). CONCLUSION: Alternative, rapid methods for DST are cost-effective and should be considered for use by national tuberculosis programs in middle-income countries.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Proteínas Bacterianas/genética , Colorimetría , Análisis Costo-Beneficio , ARN Polimerasas Dirigidas por ADN , Amplificación de Genes , Humanos , Renta/clasificación , Pruebas de Sensibilidad Microbiana/economía , Pruebas de Sensibilidad Microbiana/métodos , Micobacteriófagos/fisiología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Hibridación de Ácido Nucleico , Perú , Años de Vida Ajustados por Calidad de Vida , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Factores de Tiempo , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/epidemiología , Tuberculosis Pulmonar/microbiologíaRESUMEN
Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97,8 percent and 100 percent respectively. Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem(AU)
Asunto(s)
Humanos , Antibióticos Antituberculosos/farmacología , Farmacorresistencia Bacteriana , Micobacteriófagos/fisiología , Mycobacterium tuberculosis , Mycobacterium tuberculosis/virología , Rifampin/farmacologíaRESUMEN
BACKGROUND: Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. METHODS: Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. RESULTS: Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. CONCLUSION: Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Farmacorresistencia Bacteriana , Micobacteriófagos/fisiología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/virología , Rifampin/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Fenotipo , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Rapid, accurate and inexpensive methods are essential to detect drug-resistant Mycobacterium tuberculosis and allow timely application of effective treatment and precautions to prevent transmission. The proportion method, the MTT and Alamar Blue redox methods, and the D29 mycobacteriophage assay, were compared for their ability to detect resistance to isoniazid and rifampicin. When tested against a panel of known M. tuberculosis strains, the redox methods and the D29 assay showed good sensitivity and specificity compared to the proportion method, suggesting that they could be useful alternatives for identifying multidrug resistance in M. tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Costos y Análisis de Costo , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana/economía , Micobacteriófagos/aislamiento & purificación , Micobacteriófagos/fisiología , Oxazinas , Oxidación-Reducción , Sensibilidad y Especificidad , Sales de Tetrazolio , Tiazoles , XantenosRESUMEN
An in-house mycobacteriophage amplification assay for detecting rifampin-resistant Mycobacterium tuberculosis showed 100% sensitivity, 97.7% specificity, and 95.2% predictive value for resistance in a test of 129 isolates from a hot spot area of multidrug-resistant M. tuberculosis. The applicability of the test was demonstrated in the routine work flow of a low-resource reference laboratory.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Farmacorresistencia Bacteriana , Micobacteriófagos/fisiología , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/virología , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.