Bactericidal Activity of Liposomal Form of Lytic Mycobacteriophage D29 in Cell Models of Tuberculosis Infection In Vitro.

The use of lytic mycobacteriophages to treat tuberculosis under conditions of acquired resistance to anti-tuberculosis drugs is one of the most practical ways to improve the effectiveness of therapy and reduce the spread of this disease. We studied the efficacy of antimycobacterial action of mycobacteriophage D29 encapsulated into 400-nm liposomes in cell models of tuberculosis infection in vitro. The antimycobacterial action of lytic mycobacteriophage D29 used in free or liposome-encapsulated forms was demonstrated on cell models of intracellularly infected RAW264.7 macrophages and tuberculous granuloma formed by human blood mononuclear cells. The experiments demonstrated pronounced advantage of liposomal form of mycobacteriophage according to the criteria of their penetration into macrophages and lysis of Mycobacterium tuberculosis in culture.

Fluoromycobacteriophages for Drug Susceptibility Testing (DST) of Mycobacteria.

Fluoromycobacteriophages are a new class of reporter phages that contain Laboratorio fluorescent reporter genes (gfp, ZsYellow, and mCherry) and provide a simple means of revealing the metabolic state of mycobacterial cells and therefore their response to antibiotics. Here we described a simple and rapid method for drug susceptibility testing (DST) of Mycobacterium spp using a fluorescence microscope, a flow cytometer, or a fluorimeter in a convenient multiwell format.

Use of mycobacteriophage quantitative PCR on MGIT broths for a rapid tuberculosis antibiogram.

Phenotypic culture-based drug susceptibility testing (DST) for Mycobacterium tuberculosis is a valuable tool to identify four to six active drugs for individualized multidrug-resistant (MDR) tuberculosis (TB) regimens. Current culture-based methods are slow, however; therefore, we evaluated a rapid mycobacteriophage-based quantitative PCR (qPCR) assay for use directly on M. tuberculosis-positive MGIT broths. We compared phage qPCRs, using a simple cutoff of 3 for the ΔCq value (where Cq is quantification cycle, and ΔCq is calculated as the Cq of starting phage minus the Cq of TB isolates in drug-containing medium), on 325 clinical M. tuberculosis MGIT broth cultures versus the respective subcultured isolates tested by agar proportion. The median accuracy for the 13 drugs/concentrations tested was 98%, with most discrepancies being false-resistant results. Evaluation of phage qPCR on greater numbers of resistant strains of 393 isolates grown on Löwenstein-Jensen medium showed similar findings, with a median accuracy, sensitivity, and specificity of 97%, 90%, and 99%, respectively. This rapid culture-based DST methodology can be performed for any drug on TB-positive MGIT broths, with a specimen-to-antibiogram turnaround time of approximately 23.9 days, compared with waiting 58.6 days for isolate growth on solid medium followed by agar proportion DST.

A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6.

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various gram-positive bacteria and mycobacteria.

Comparison of the performance of two mycobacteriophage D29-based protocols for fluoroquinolone susceptibility testing in Mycobacterium tuberculosis.

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.

Comparison of the performances of two in-house rapid methods for antitubercular drug susceptibility testing.

Resistance to rifampin (rifampicin), isoniazid, and streptomycin of 69 Mycobacterium tuberculosis isolates was analyzed by an in-house method based on mycobacteriophage D29 and a colorimetric micromethod. Both methods showed sensitivity and specificity values ranging from 93% to 100%. These simple methods offer an option for drug resistance assessment of M. tuberculosis.

[Rapid rifampicin susceptibility test by using recombinant mycobacteriophages].

OBJECTIVE: To set up a fast, sensitive and specific way to detect mycobacteria and rifampicin susceptibility to mycobacteria by using recombinant mycobacteriophages and bioluminescent method. METHODS: Firstly detected the luciferase activity in different bacteria by using mycobacteriophages, then assessed the best drug concentration of rifampicin for drug susceptibility test in luciferase reporter assay, and lastly used the above conditions to decide rifampicin susceptibilities to different mycobacteria and compare the result with L-J medium culture. RESULTS: Bacteriophages had high light production specifically in mycobacteria and only very low light production in E. coli, the difference being obvious. The light production of rifampicin-resisitant mycobacterium strains was much higher than that of sensitive strains in culture with rifampicin(P < 0.05). Different drug concentrations of rifampicin were used to optimize drug concentration for rifampicin drug susceptibility test and the optimum concentration was found to be 2 micrograms/ml. The correlation of drug susceptibility test between luciferase reporter phages to L-J medium in standard strains and clinical isolates was the sarce. Temperature sensitive Phage 88 was more sensitive than Phage 40(P < 0.05). The time for drug resistance test was 72 hours. CONCLUSIONS: Luciferase reporter phage can detect mycobacteria specifically and can be used in rifampicin susceptibility test. This assay is a fast, sensitive and specific method to detect mycobacterium strains and their resistance to rifampicin.

TB: the return of the phage. A review of fifty years of mycobacteriophage research.

The first mycobacteriophage was isolated in 1947, and since that time over 250 of these viruses have been identified. Phages have made a significant contribution to our knowledge of mycobacteria over the past fifty years, and following the development of typing techniques in the 1960s and 1970s they were widely used in epidemiological studies of tuberculosis. Unfortunately, attempts to use lytic phages therapeutically during tuberculosis infection have so far failed to elicit cure in experimentally infected animals. During the past decade phages have become important in molecular studies of mycobacteria, both in terms of studying phage biology and as tools in recombinant DNA technology, thus facilitating the investigation of mycobacterial pathogenesis. Today their potential as diagnostics reagents is also being realised with the development of exciting new techniques for rapid bacterial detection and drug susceptibility testing. This review outlines the history of these remarkable organisms, from their discovery fifty years ago to the current developments in rapid diagnostic techniques.

Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis.

There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.

Action of colistin (polymyxin E) on the lytic cycle of the mycobacteriophage D29 in Mycobacterium tuberculosis.

The antibiotic colistin (polymyxin E) inhibited the lytic cycle of the mycobacteriophage D29 in the tubercle bacilli, but not the D29 adsorption. The protein and nucleic acid synthesis in D29-infected bacteria were not affected significantly. The inhibitory activity was reversed by washing off the antibiotic, and by addition of Ca++, but not in media made iso-osmotic by addition of NaCl or sucrose. Transmission electron microscopy revealed an asymmetric to symmetric transition in the staining profile of the cytoplasmic membrane. Though no mature phage particles were ever observed in colistin-treated, D29-infected tubercle bacilli, loosely arranged aggregates resembling phage proheads were occasionally found. Judging from the above data, it was concluded that colistin inhibited D29 lytic cycle by causing molecular displacements in the inner leaflet of the cytoplasmic membrane, and consequently, the binding sites for D29 structural proteins were not available.

Effect of heat treatment on phage susceptibility of a Mycobacterium smegmatis strain.

Plating efficiency of mycobacteriophage butyricum (By) proved to be 10(-4)-10(-3) on Mycobacterium smegmatis strain Rabinowitz (M. sm. R.) cells being in the logarithmic phase. This increased to 10(-2)-10(-1) when the cells were held at 50-57 degrees C for 2 h before infection. After replacing the cells to 37 degrees C, plating efficiency of the phages returned to the starting values. This phenomenon could be inhibited by nalidixic acid (150 micrograms/ml) and chloramphenicol (10 micrograms/ml) but not by mitomycin C (0.05 microgram/ml). No return to the starting plating efficiency values were observed, if incubation of cells at 37 degrees C after heat treatment has been performed in buffer. The data suggest that By phage propagation in M. sm. R. cells is inhibited by a thermosensitive protein.

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA.

Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl2, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From 32P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).

[Comparative study of the chemical composition and properties of the capsule polysaccharides in M-, S- and R-variants of Mycobacterium lacticolum]. / Sravnitel'noe izuchenie khimicheskogo sostava i nekotorykh svoistv polisakharidov kapsul M-, S- i R-variantov Mycobacterium lacticolum.

Polysaccharides from the capsules of Mycobacterium lacticolum M, S and R variants were comparatively studied for the first time. The polysaccharides from M and S cells contained galactose, glucose and mannose; however, the polysaccharides must be different since they vary in specific rotation. The capsule polysaccharide from R cells contained also arabinose. Its specific rotation differed considerably from those of M and S cells. The polysaccharides are involved in phage adsorption on mycobacterial cells, and the three variants show differences in this respect. The free exopolysaccharides of M, S and R variants are identical with the corresponding capsule exoglycans, and their proportions differ among the variants.

Effects of antituberculosis and antileprosy drugs on mycobacteriophage D29 growth.

The minimal inhibitory concentrations of antituberculosis and antileprosy drugs were determined for Mycobacterium aurum. The concentrations that reduced the final yield of bacteriophage D29R1 by 50% and the time during the replication cycle at which the drugs completely inhibited phage production were estimated. THe 50% inhibitory concentration/minimal inhibitory concentration ratios were close to 1.0 for clofazimine, colistin, rifampin, and streptomycin; these ratios were high for dapsone (diaminodiphenylsulfone) and isoniazid. Ethambutol (minimal inhibitory concentration, 1.0 micrograms/ml) was without effect on viral growth.

Requirement for calcium ions in mycobacteriophage I3 DNA injection and propagation.

Ca2+ ions are absolutely necessary for the propagation of mycobacteriophage I3 in synthetic medium. These ions are required for successful infection of the host and during the entire span of the intracellular development of the phage. A direct assay of the phage DNA injection using 32[P] labelled phage, shows that Ca2+ ions are necessary for the injection process. The injection itself is a slow process and takes 15 min to complete at 37 degrees C. The bacteria infected in presence of Ca2+ tend to abort if the ions are subsequently withdrawn from the growth medium. The effect of calcium withdrawal is maximally felt during the early part of the latent period; however, later supplementation of Ca2+ ions salvage phage production and the mature phage progeny appear after a delayed interval, proportional to the time of addition of Ca2+.

On the mechanism of the antimycobacterial activity of isoniazid + prothionamide + dapsone (Isoprodian).

Increased antimycobacterial activity of Isoprodian (isoniazid + prothionamide + dapsone) may be due to (i) decreased mutation rate for INH resistance provoked in mycobacteria by DDS (in 2 of 3 strains tested); (ii) leakage of K+, Na+ or Ca++ caused by INH and/or by PTH (in all 3 strains tested), and (iii) indicating damages which may increase the low level penetration of DDS into the cell in sub-MIC concentrations as shown by phage proliferation changes (tested in 1 strain with 1 phage).