Exposure to cetyl pyridinium chloride and loss of integrity of cell wall of mycobacteria.

Background: Cetyl pyridinium chloride (CPC) liquefied sputum was shown to reduce AFB smear positivity presumably damaging cell wall of M. tuberculosis. Settings: National Institute for Research in Tuberculosis, Chennai, (Tamil Nadu). Objective: To assess the cell wall damage of mycobacteria in CPC liquefied sputum, by Transmission Electron Microscopy (TEM) and mycobacteriophage adsorption studies. Methods: Pooled sputum sample from smear positive pulmonary TB patients was homogenized and liquefied with CPC. It was examined in TEM daily for four days, to assess cell wall damage of M. tuberculosis, and photomicrographs were taken. M. smegmatis mc2155, treated with CPC, was infected with mycobacteriophage (phAE129) to study phage adsorption on cell wall and plaque formation. CPC untreated sputum and M. smegmatis formed controls. Results: Photomicrographs showed that cell wall of M. tuberculosis was intact in controls and damaged in CPC preserved sputum for 96 hours. Plaque formation was seen and absent respectively in CPC untreated and treated M. smegmatis cells. Conclusion: Exposure to CPC damaged the cell wall of M. tuberculosis within 96 hours. Mycobacteriophage failed to form plaques after M. smegmatis mc2155 was treated with CPC implying inhibition of phage adsorption on damaged cell wall and thus providing a clue for poor staining and smear positivity in microscopy.

Luciferase reporter phage phAE85 for rapid detection of rifampicin resistance in clinical isolates of Mycobacterium tuberculosis

OBJECTIVE@#To evaluate luciferase reporter phage (LRP) phAE85 in rapid detection of rifampicin resistance in a region where TB is endemic.@*METHODS@#One hundred and ninety primary isolates on Lowenstein-Jensen medium were tested. Middlebrook 7H9 complete medium with and without rifampicin at 2 μg/mL was inoculated with standard inoculum from suspensions of the clinical isolate. After incubation for 72 h, LRP was added. Following 4 h of further incubation, light output from both control and test was measured as relative light units. Strains exhibiting a reduction of less than 50% relative light units in the drug containing vial compared to control were classified as resistant. Results were compared with the conventional minimum inhibitory concentration method (MIC) of drug susceptibility testing.@*RESULTS@#The two methods showed high level of agreement of 97% (CI 0.94, 0.99) and P value was 0.000 1. The sensitivity and specificity of LRP assay for detection of rifampicin resistance were 91% (CI 0.75, 0.98) and 99% (CI 0.95, 1.00) respectively. Time to detection of resistance by LRP assay was 3 d in comparison with 28 d by the minimum inhibitory concentration method.@*CONCLUSIONS@#LRP assay with phAE85 is 99% specific, 91% sensitive and is highly reproducible. Thus the assay offers a simple procedure for drug sensitivity testing, within the scope of semi-automation.

In-house, simple & economical phage technique for rapid detection of rifampicin, isoniazid, ethambutol, streptomycin & ciprofloxacin drug resistance using Mycobacterium tuberculosis isolates.

Background & objectives: Multiple drug resistance (MDR) among Mycobacterium tuberculosis poses a serious therapeutic problem. Early detection of MDR can be valuable but the conventional drug susceptibility tests take 4-6 wk time after the laboratory isolation of M. tuberculosis. The bacterial phage assay has been reported as a rapid tool for rifampicin susceptibility testing of tubercle bacilli using the suspension of isolated cultures. The present study was aimed to set up a phage assay for testing drug susceptibility to isoniazid (INH), rifampicin, ethambutol, streptomycin and ciprofloxacin in M. tuberculosis isolates. Methods: Mueller-Hinton broth instead of Middle Brook 7H9 broth was used to make it more economical. The phage assay was compared with the proportion method using 100 M. tuberculosis isolates from pulmonery TB cases. Phage assay results were available in 48 h for rifampicin and streptomycin while 72 h required for INH, ethambutol and ciprofloxacin. The assay was compared with gold standard proportion method. Interpretation of the results was easy and clear. Results: In the present study, sensitivity and specificity of the phage assay when compared to proportion method were in the range of 97 to 100 per cent for all the drugs except for ciprofloxacin for which it was 93 and 96 per cent, respectively. Interpretation & conclusions: The phage assay was economic, easy to perform and rapid for the detection of drug resistance in M. tuberculosis isolates with no requirement of expensive equipment. It is within the reach of microbiology laboratories in developing countries having high loads of tuberculosis.

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.

Expression and purification of lysin B in mycobacteriophage D29 and analysis of its enzymatic properties / 生物工程学报

LysinB (LysB) in mycobacteriophage D29 was cloned and expressed and its enzymatic properties were analysed. The lysB gene was amplified by PCR from mycobacteriophage D29 genomic DNA and inserted into pET22b vector. The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3) to express fusion protein, which was purified by Ni-NTA column and enzymatic activity detected. The results showed that expression plasmid pET22b-lysB was constructed successfully. Highly purified recombination protein (His-LysB) was obtained 33.2 mg from 1 L LB culture medium. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. With p-nitrophenyl butyrate as substrate, the thermal stability of the enzyme was poor when the temperature was above 30 degrees C. The enzyme exhibited higher stability at pH 5.0-9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23 degrees C and 7.5 respectively. Under the optimum conditions, the specific activity of His-LysB was 1.3 U/mg. Zn2+, CU2+, Mg2+, Mn2+ and phenylmethane sulfonyl fruoride severely inhibited the lipolytic activity of His-LysB. The result provides a new option for tuberculosis drug research and development.

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.

Establishment and evaluation of nitrate reductase combined with mycobacteriophage assay / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a rapid, inexpensive, and simple drug susceptibility test (DST) for Mycobacterium tuberculosis (M. tb) and evaluate its feasibility.</p><p><b>METHOD</b>We used nitrate reductase combined with mycobacteriophage assay (PhaB-NRA) to test 49 clinical M. tb isolates of, and the results were compared with those of PhaB-NRA and traditional absolute concentration method.</p><p><b>RESULTS</b>The sensitivity, specificity, and accuracy of PhaB-NRA for rifampicin were 89.1%, 91.67%, and 89.8%; on the contrary, those of isonicotinyl hydrazide were 86.21%, 90.0%, and 87.8%, respectively. The coincidence between PhaB-NRA and traditional assay were 0.746 for rifampicin and 0.750 for isonicotinyl hydrazide.</p><p><b>CONCLUSIONS</b>PhaB-NRA is an inexpensive, rapid, and simple DST method. It is a promising rapid screening technique for DST of M. tb.</p>

Evaluation of the usefulness of phage amplification technology in the diagnosis of patients with paucibacillary tuberculosis.

The present study was undertaken to assess the performance of the Fast Plaque TB(TM) (FPTB) test in the diagnostically difficult group of paucibacillary tuberculosis (TB) and to compare its results with the conventional bacteriological methods. The study was conducted on a total of 139 patients, who were negative for TB in sputum-smear examination. Bronchoalveolar lavage (BAL) or pleural biopsy specimens collected from these patients were subjected to smear examination, LJ culture and FPTB test. The smear, culture and the FPTB positivity rates were compared between patients with pulmonary and pleuro-pulmonary involvement. The FPTB test was found to register an overall sensitivity of 58.8% and specificity of 97.9%. The positive and negative predictive values of the test were 98.1 and 56.5, respectively. Among patients with paucibacillary TB, on head-to-head comparison, we found that the sensitivity and specificity values of the FPTB test were marginally better than smear-microscopy and inferior to culture on LJ media.

Evaluation of a microcolony detection method and phage assay for rapid detection of Mycobacterium tuberculosis in sputum samples.

Early and rapid diagnosis of tuberculosis is necessary for both treatment and control of the disease. This study evaluated two microcolony observation techniques based on liquid and solid media and a mycobacteriophage assay, to evaluate their effectiveness in the diagnosis of pulmonary TB compared with a standard culture (BACTEC 460 and LJ medium). Middlebrook7H9 (M7H9) broth based on microcolony determination detected 57/61 positives cultures (n = 200) with a sensitivity of 93.4% and a specificity of 87.1%. M7H11 agar detected 57/62 positive cultures (n = 198) with a sensitivity of 91.9% and a specificity of 89.7%. The mycobacteriophage assay detected 98/143 (68.5%) of positive samples. The time to positivity was 48 hours in the mycobacteriophage assay versus 7 days in both the M7H9 broth and M7H11 agar. The costs in comparison with the culture (BACTEC 460 and LJ) were 33% and 48% for the microcolony and mycobacteriophage methods, respectively. Microcolony methods were rapid and cost effective compared to standard cultures. The mycobacteriophage assay, despite its lower sensitivity, has a short turn around time, and may be recommended as a screening test in countries with a low prevalence of tuberculosis.

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.

Rapid detection of rifampicin susceptibility of mycobacterium tuberculosis in sputum specimens by mycobacteriophage assay

To evaluate the performance of FASTPlaqueTB-RIF TM, a newly introduced bacteriophage assay for rapid detection of rifampicin susceptibility of Mycobacterium tuberculosis in sputum specimens. A comparative study of 40 sputum specimens from patients of pulmonary tuberculosis, using FASTPlaqueTB-RIF TM and Bactec 460 TB system carried out at the Armed Forces Institute of Pathology, Rawalpindi between September and November 2001. Of the 40 clinical isolates of Mycobacterium tuberculosis tested for rifampicin [RIF] susceptibility using the Bactec 460 TB system, 28 isolates were resistant to RIF and 12 isolates were susceptible. FASTPlaqueTB-RIF TM identified 24 specimens as resistant to RIF. Three specimens that revealed susceptible isolates on Bactec 460, were resistant by FASTPlaqueTB-RIF' while four specimens which revealed resistant isolates on Bactec 460, demonstrated susceptibility to RIF by FASTPlaqueTB-RIF TM. The sensitivity and specificity of FASTPlaqueTB-RIF TM were 86% and 73% respectively. The predictive values of positive and negative tests were 0.89 and 0.67 respectively. The overall accuracy of the technique was 82%. The phage assay took 48 hours to perform. Early detection of rifampicin resistance by the mycobacteriophage technique direct from sputum specimens is a potentially useful new test which would allow decision regarding appropriate therapy to be made early thus having a positive impact on patient care and on prevention of spread of MDR TB

update on the diagnosis of tuberculosis

Tuberculosis [TB] continues to be the bane of mankind. Early diagnosis is the cornerstone of tuberculosis control strategies. Recent years have seen major advances in the fields of biotechnology and molecular biology with introduction of several new diagnostic techniques for tuberculosis and improvement in the existing ones. The new automated culture techniques have appreciably reduced the time required for detection and antimicrobial susceptibility testing. The molecular amplification techniques like the Polymerase Chain Reaction [PCR] have made the same-day diagnosis a reality. Improvements in serology and introduction of novel new techniques like the bacteriophage assays have also shown a lot of promise. However, most of these new techniques are too expensive and sophisticated to be of any practical benefit to the vast majority of TB patients living in underdeveloped countries like Pakistan for whom an early and inexpensive diagnosis remains as elusive as ever. In this article various existing modalities as well as the new advances in TB diagnostics are reviewed

Cloning and characterization of mycobacteriophage I3 promoters.

Restriction fragments of mycobacteriophage I3 DNA capable of initiating transcription have been cloned into a promoter selection vector of Escherichia coli, and selected on the basis of development of resistance to chloramphenicol. The growth pattern of these 'promoter clones' on a concentration gradient of chloramphenicol and the biochemical assays of the chloramphenicol acetyl transferase have permitted the assessment of their relative promoter strengths. DNA sequence analysis revealed significant homology of these promoters to the -35 regions of the mycobacterial--and E. coli promoter consensus, but less so the -10 region. Based on the sequence of phage I3 promoters identified here and the reported sequences of mycobacterial promoters, a promoter consensus for mycobacteria has been generated.