Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose / Development of recombinant micobacteriophage for rapid detection of tubercle bacillus

O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula.

Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.

Mycobacteriophages as versatile tools for genetic manipulation of mycobacteria and development of simple methods for diagnosis of mycobacterial diseases / Micobacteriófagos como herramientas versátiles para la manipulación genética y el desarrollo de métodos simples para el diagnóstico de enfermedades micobacterianas

Tuberculosis, caused by Mycobacterium tuberculosis, is responsible for over two million deaths per year worldwide. Due to its long doubling time (18 h), the microbiological detection of M. tuberculosis by conventional methods takes up to one month, unless the number of bacilli in the biological sample is high enough. Thus, drug resistance assessment requires at least one month for obtaining the primary culture and another month to determine its susceptibility to antimycobacterial drugs. Moreover, for a long time, the lack of genetic tools for mycobacteria has been a barrier for undertaking studies aimed at understanding the mechanisms of drug resistance and drug target identification, being all these topics of utmost importance considering the increase in the number of drug-resistant clones and the few therapeutic options available. Mycobacteriophages are promising as a novel source of genetic elements for mycobacteria manipulation, as well as for the development of versatile, simple, fast and cheap methods for drug resistance assessment of M. tuberculosis clinical isolates. We herein describe the background related to the use of mycobacteriophages, with emphasis placed on their utilization for drug resistance analysis in our country.

La tuberculosis, enfermedad causada por el bacilo Mycobacterium tuberculosis, es responsable de más de dos millones de muertes anuales en el mundo. Debido a su largo tiempo de duplicación (18 h), la detección bacteriológica de M. tuberculosis por métodos convencionales necesita de un mes o aun más, a menos que el número de bacilos en la muestra clínica sea suficientemente alto. Por consiguiente, se necesita un mínimo de dos meses para determinar la resistencia de este microorganismo a las drogas antituberculosas: uno para obtener el cultivo primario y otro para ensayar la sensibilidad frente a aquellas. La falta de herramientas para la manipulación genética de micobacterias ha dificultado la identificación de los blancos de acción de las drogas y el estudio de los mecanismos de resistencia a éstas, tópicos de la mayor relevancia dado el aumento mundial del número de aislamientos clínicos multirresistentes y las pocas opciones terapéuticas disponibles. Los micobacteriófagos son considerados nuevas herramientas para la manipulación de las micobacterias, así como para el desarrollo de métodos simples, rápidos y económicos para determinar la sensibilidad a drogas de los aislamientos clínicos de M. tuberculosis. En esta revisión se describen los antecedentes del uso de micobacteriófagos con énfasis en su utilización para el análisis de resistencia a drogas antituberculosas en nuestro país.

Recent advances in molecular methods for early diagnosis of tuberculosis and drug-resistant tuberculosis

Tuberculosis (TB) remains the main infectious cause of deaths in the world. Due to the slow metabolism of the causative agent, Mycobacterium tuberculosis, the isolation, identification and drug susceptibility testing requires several weeks. New techniques have improved specificity, turnaround time and cost effectiveness. Although these methods yield results within hours from sample collection, the clinical significance of each positive result requires rigorous evaluation in most cases. Herein the advantages and disadvantages of the most promising molecular techniques for detection of TB and drug resistance are discussed.

Avances recientes en métodos moleculares para el diagnóstico precoz y tuberculosis resistente al tratamiento La tuberculosis sigue siendo la principal causa de mortalidad por un agente infeccioso a escala mundial. Debido al metabolismo lento de su agente etiológico, Mycobacterium tuberculosis, el aislamiento, la identificación y las pruebas de susceptibilidad tardan varias semanas. Nuevas técnicas moleculares desarrolladas ofrecen mejorías en la especificidad, el tiempo para la obtención de resultados y su costo-efectividad. Estas pruebas producen resultados en pocas horas a partir de la toma de muestra, pero su relevancia clínica requiere aún ser evaluada rigurosamente en la mayoría de los casos. En esta revisión se discuten las ventajas y las desventajas de las pruebas moleculares más promisorias desarrolladas para el diagnóstico de la tuberculosis y la tuberculosis resistente a medicamentos.