Growth Of Tubercle Bacilli Research Articles

Naphthalimides Selectively Inhibit the Activity of Bacterial, Replicative DNA Ligases and Display Bactericidal Effects against Tubercle Bacilli.

The DNA ligases, enzymes that seal breaks in the backbones of DNA, are essential for all organisms, however bacterial ligases essential for DNA replication use β-nicotinamide adenine dinucleotide as their co-factor, whereas those that are essential in eukaryotes and viruses use adenosine-5′-triphosphate. This fact leads to the conclusion that NAD+-dependent DNA ligases in bacteria could be targeted by their co-factor specific inhibitors. The development of novel alternative medical strategies, including new drugs, are a top priority focus areas for tuberculosis research due to an increase in the number of multi-drug resistant as well as totally drug resistant tubercle bacilli strains. Here, through the use of a virtual high-throughput screen and manual inspection of the top 200 records, 23 compounds were selected for in vitro studies. The selected compounds were evaluated in respect to their Mycobacterium tuberculosis NAD+ DNA ligase inhibitory effect by a newly developed assay based on Genetic Analyzer 3500 Sequencer. The most effective agents (e.g., pinafide, mitonafide) inhibited the activity of M. tuberculosis NAD+-dependent DNA ligase A at concentrations of 50 µM. At the same time, the ATP-dependent (phage) DNA LigT4 was unaffected by the agents at concentrations up to 2 mM. The selected compounds appeared to also be active against actively growing tubercle bacilli in concentrations as low as 15 µM.

Exit from mycobacterial dormancy: a structural perspective.

Dormancy in mycobacteria is defined as a stable but reversible non-replicating state in response to stresses. In Mycobacterium tuberculosis, an important human pathogen, this state is responsible for latent Tuberculosis. The current chemotherapy to defeat Tuberculosis while effective in killing growing tubercle bacilli is largely ineffective in killing dormant bacilli. For this reason there is a recent interest to develop new drugs against this disease in the latent form. To this aim, the knowledge of the molecular basis of bacterial resuscitation from dormancy is necessary and of paramount importance. This review summarizes the current knowledge on the complex mechanism of exit from mycobacterial dormancy; the main molecular players responsible for mycobacterial resuscitation from dormancy are described and their role is discussed from a structural perspective.

Diagnostic luciferase reporter phage assay for active and non-replicating persistors to detect tubercle bacilli from sputum samples

Diagnosis of latent tuberculosis infection is a myth for want of a simple, direct tool. Simulation of hypoxic environment was done to create a novel hypothetical model for persistence using processed sputum samples. The adaptation of tubercle bacilli to hypoxic environment seems to be influenced by pre-existing clinical status of the patients at the time of sputum collection, resulting in varied growth pattern. Bacilli from 36 samples did not get adapted to latency of which 15 samples were from patients in whom the disease was well established and the tubercle bacilli in them probably did not experience any stress whatsoever. Similarly, 10 of the 37 samples showing the presence of cultivable cells in both aerobic and anaerobic conditions were from patients who had relapsed. The bacilli in these samples had been probably experiencing stress and thus were ready to adapt to the hypoxic environment. Diagnostic luciferase reporter phage assay for non-replicating persistors (DLRPA-NRP) identified 30 additional positives which failed to grow on Lowenstein–Jensen medium. Presence of viable bacilli in these samples was confirmed by reverse transcriptase-PCR (RT-PCR) for 16S rRNA indicating either the improved sensitivity of the assay to detect actively growing bacilli or its ability to detect non-replicating persistors. The utility of LRP assay to detect both dormant and active tubercle bacilli was explored in this work and was optimized using lysis inhibition to diagnose tuberculosis with rapidity, improved sensitivity and specificity. DLRPA-NRP, a rapid growth based assay is thus developed to detect both dormant and actively growing tubercle bacilli.

Cytological and Transcript Analyses Reveal Fat and Lazy Persister-Like Bacilli in Tuberculous Sputum

BackgroundTuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis.Methods and FindingsMicroscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body–positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body–positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R2 = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population.ConclusionAs nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.

The resumption of consumption: a review on tuberculosis

Among all infectious diseases that afflict humans, tuberculosis (TB) remains the deadliest. At present, epidemiologists estimate that one-third of the world population is infected with tubercle bacilli, which is responsible for 8 to 10 million new cases of TB and 3 million deaths annually throughout the world. Approximately 95% of new cases and 98% of deaths occur in developing nations, generally due to the few resources available to ensure proper treatment and where human immunodeficiency virus (HIV) infections are common. In 1882, Dr Robert Koch identified an acid-fast bacterium, Mycobacterium tuberculosis, as the causative agent of TB. Thirty-nine years later, BCG vaccine was introduced for human use, and became the most widely used prophylactic strategy to fight TB in the world. The discovery of the properties of first-line antimycobacterial drugs in the past century yielded effective chemotherapies, which considerably decreased TB mortality rates worldwide. The later introduction of some additional drugs to the arsenal used to treat TB seemed to provide an adequate number of effective antimicrobial agents. The modern, standard short-course therapy for TB recommended by the World Health Organization is based on a four-drug regimen that must be strictly followed to prevent drug resistance acquisition, and relies on direct observation of patient compliance to ensure effective treatment. Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents due to the low permeability of its cell wall. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance. M. tuberculosis mutants resistant to any single drug are naturally present in any large bacterial population, irrespective of exposure to drugs. The frequency of mutants resistant to rifampicin and isoniazid, the two principal antimycobacterial drugs currently in use, is relatively high and, therefore, the large extra-cellular population of actively metabolizing and rapidly growing tubercle bacilli in cavitary lesions will contain organisms which are resistant to a single drug. Consequently, monotherapy or improperly administered two-drug therapies will select for drug-resistant mutants that may lead to drug resistance in the entire bacterial population. Thereby, despite the availability of effective chemotherapy and the moderately protective vaccine, new anti-TB agents are urgently needed to decrease the global incidence of TB. The resumption of TB, mainly caused by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains and HIV epidemics, led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. The latter should be effective to combat both drug-susceptible and MDR/XDR-TB.

The immune response to mycobacterial infection: Development of new vaccines

Pulmonary tuberculosis continues to fluorish worldwide despite our most vigorous attempts to control it. After nearly a century of study we still know very little about the virulence factors of M. tuberculosis or M. bovis or how they trigger the protective immune response within the infected host. This anti-tuberculosis response is mediated by a population of specifically sensitised T lymphocytes which activate the monocytes entering the developing lesion from the bloodstream. The immunologically activated macrophage induces a persistent bacteriostasis which is usually sufficient to protect the host although it will not eliminate the infection altogether so that reactivation can occur whenever the cellular defences are depleted as a result of aging or immunosuppressive chemotherapy. Protective immunogens released by actively growing tubercle bacilli give rise to a protective cell-mediated, rather than a humoral (non-protective) immunity. The genes responsible for the production of these “protective” antigens are being cloned and transferred to suitable myobacterial vector by means of the newly developed “shuttle phasmid”. Development of such recombinants constitute the first step in preparing more effective anti-tuberculous vaccines for future use against these important human and animal pathogens.

A method of detecting tubercle bacilli in sputum

Tubercle bacilli were injected intravenously to mice after dusts were injected into their subcutaneous tissue. Out of 23 kinds of dusts tested, a quartz dust enhanced most the growth of mycobacteria injected intravenously in mice. Based on this a mixture of the sputum from patients with pulmonary tuberculosis and finely powdered crystalline silica was inoculated subcutaneously into the flanks of mice. Growth of tubercle bacilli was demonstrated occasionally for the sputum specimens from smear-positive and culture-negative cases, and even for smear- and culture negative cases. When a sputum specimen which yielded many colonies on routine egg medium were tested, the growth of bacteria became visible as early as 2 weeks after inoculation. On the other hand, it took 3 to 4 weeks for the visible growth of bacilli, when a sputum specimen yielded only a few or no colonies on culture medium. In this method, the bacilli appeared earlier and more frequently than in the routine culture method on egg medium. Thus an inoculation into mice with a mixture of the sputum and quartz powder was shown to be a sensitive method for the diagnosis of pulmonary tuberculosis.

Tuberculostatic activity of henna ( Lawsonia inermis Linn.)

The tuberculostatic activity of the herb henna ( Lawsonia inermis Linn.) was tested in vitro and in vivo. On Lowenstein Jensen medium, the growth of tubercle bacilli from sputum and of Mycobacterium tuberculosis H 3 7Rv was inhibited by 6 μg/ml of the herb. In vivo studies on guinea pigs and mice showed that the herb at a dose of 5 mg/kg body weight led to significant resolution of experimental tuberculosis following infection with M. tuberculosis H 3 7Rv.

Effects of lysolecithin on the growth of tubercle bacilli in vitro

Effects of lysolecithin on the growth of tubercle bacilli in vitro

Inhibitory Effect in Vitro of Commercial Products of Antituberculosis Agents on the Growth of Tubercle Bacilli

Inhibitory Effect in Vitro of Commercial Products of Antituberculosis Agents on the Growth of Tubercle Bacilli

In VitroStudies on the Mechanism of Acquired Resistance to Tuberculous Infection

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.

Role of mycobactin in the growth and virulence of tubercle bacilli.

Tubercle bacilli failed to grow in iron-void media enriched with solutions of iron-containing transferrin (Tr) or ferritin (F) because these substances do not provide the bacilli with iron, which is essential for their growth. Animal serum and macrophages possessed no iron carrier with an ability to satisfy the need of the bacteria for the metal. Mycobactin (M), the growth-product of tubercle bacilli, removed iron from Tr and F and supplied the metal for bacillary utilization. The role of M in the growth of tubercle bacilli was influenced by nonionic surfactants which inhibited bacillary growth by removing M from the bacillary cells and interfering with the absorption of M-iron complexes. Experiments with Tween 80, Triton WR-1339, and lecithin showed that avirulent bacilli lose M at lower concentrations of the surfactants than virulent bacilli. Since avirulent and virulent bacilli possess the same amount of M, these findings indicate that M is bound more firmly to lipid-rich virulent than lipid-poor avirulent cells. These findings indicate that the resistance of virulent bacilli to the M-removing activity of the surfactants is an indicator of their ability to multiply in the infected host and may be used as a measure of bacillary virulence.

Effects of normal and activated cell fractions on the growth of tubercle bacilli.

Fractions prepared from normal and activated liver cells were tested for the antimycobacterial activity by the agar plate diffusion test. Results showed that lysosome extracts of normal and activated cells exerted no antibacterial activity, cell extracts and lysosome membranes exerted some activity, and cell membranes exerted the strongest activity. The active materials of activated cells exerted stronger antituberculous activity than the corresponding materials of normal cells. Degrees of the antimycobacterial activity of various cell fractions showed a close correlation with the amounts of nonesterified fatty acids. This correlation, as well as other data, suggested that the antimycobacterial activity of cell fractions was caused by toxic fatty acids which were produced during the hydrolysis of lipoporteins or phospholipids by the activity of tissue lipases. The relationship of these findings to the mechanism of cellular immunity is discussed.

The growth of tubercle bacilli on nutrient agar and the biological properties of the strains subcultured on the medium

The growth of tubercle bacilli on nutrient agar and the biological properties of the strains subcultured on the medium

Mechanism of tuberculostasis in mammalian serum. II. Induction of serum tuberculostasis in guinea pigs.

The growth of tubercle bacilli in serum samples of untreated animals depends upon the availability of ionic iron which serves as a growth factor in supporting bacillary multiplication. The amount of available iron in serum is determined by the ratio between iron-saturated and iron-free transferrin; a low value for the ratio is associated with tuberculostasis (e.g., human serum, 0.4), whereas a high value is associated with the growth-supporting quality (e.g., guinea pig serum, 5.6). The treatment of guinea pigs with lipopolysaccharide of Escherichia coli or tuberculous cell wall material consistently and significantly reduced serum iron levels; a similar but less striking effect was observed in BCG-vaccinated animals. Pronounced differences were observed in the time of appearance and duration of serum hypoferremia; in lipopolysaccharide-treated animals, it appeared in 1 day and lasted for several days, whereas in BCG-vaccinated animals it appeared in about 2 weeks and lasted for much longer time periods. The induced hypoferremia was always associated with the concomitant development of serum tuberculostasis which could be neutralized by the addition of iron. These results indicate, therefore, that the mechanism of induced serum tuberculostasis in lipopolysaccharide- or tuberculous cell wall-treated and BCG-vaccinated guinea pigs is the same as that present in tuberculostatic sera of untreated animals.

In vitro effect of iron salts and chelating agents on serum tuberculostasis.

Normal human serum was found to inhibit the growth of tubercle bacilli in vitro. Ferric and ferrous ions abolished this tuberculostatic activity when concentration of the cations exceeded total iron-binding capacity of serum. Utilization of iron by tubercle bacilli was confirmed by uptake of radioactive iron. When added extraneous iron was bound by chelating agents such as human transferrin or desfernoxamine B, tubercle bacilli ceased to grow, indicating that the antimycobacterial action of serum is related to its nonspecific iron-binding capacity.

STUDIES ON MEDIA FOR CULTIVATION OF TUBERCLE BACILLI 2. Effects of temperature of inspissation and storage Ogawa's egg media and different conditions of cultivation for growth of the tubercle bacilli.

Heating for coagulation of Ogawa's egg media was started from 55℃ for evaporation of air in inside of the media and the temperature was elevated gradually about 10℃ during fifteen minutes in speed by controlling ther-mostat of electric automatic inspissator. So that, for preparation of egg solid media coagulated at 85℃ it needs about forty-fuve minutes, starting from 55℃. The authors prepared Ogawa's egg media coagulated at different temperatures by using the above processes and compared effects of different temperature of coagulation and storage of Ogawa;s media and different conditions of cultivation for growth of tubercle bacilli. Inoculated media were sealed tightly with rubber caps. 1. Optimal coagulation temperature of Ogawa's egg media for growth of tubercle bacilli was 85℃ and this temperature was more optimal than 90℃ for preparation of Ogawa's media in number of colonies and also in size of colonies. The media coagulated one hour at 90℃ was worse than the above two kinds of media for growth of tubercle bacilli. 2. There were no significant differences for growth of tubercle bacilli between Ogawa's media stored at 10℃ during four months and freshly prepared Ogawa's media. And also Ogaawa's media stored at room temperature during four months from March the July were still useful for cultivation of tubercle bacilli. It seems important for long-time storage of Ogawa's egg media to protect dryness of media as much as low temperature. 3. Tight sealing of inoculated media was better for growth of small number of tubercle bacilli, i.e. from few to several hundred viable units, than incomplete sealing of inoculated media.

STUDY ON THE TUBERCULOSTATIC FACTOR OF MAMMALIAN SERUM.

Studies of the effects of serum from various mammals upon the tubercle bacillus have generally been limited in the time of contact between the bacillus and the investigated serum, and in the use of diluting media which can neutralize tuberculostasis. It was shown in previous reports that the latter limitation is mainly responsible for failures to demonstrate a strong antimycobacterial activity in immune guinea pig and normal human serum (1, 2)when these sera were diluted in various citrateor phosphate-containing media, tuberculostasis was vitiated. The use of different media for the dilution of investigated serum is the principal cause of the contradictory results reported in the literature. Thus, Kirchner (3) and Boissevain (4) diluted human, guinea pig, sheep, rabbit, and horse serum in synthetic media and concluded that these sera stimulated the growth of human tubercle bacilli, and that such serummedium mixtures could serve as an excellent medium for the growth of these organisms. Raffel (5), in discussing immunity in tuberculosis, indicated that there is no specific evidence in the literature to suggest that the serum of normal animals is tuberculostatic-virulent as well as avirulent bacilli grow very well in vitro in normal serum as a medium. Contrary to these findings, Hedvall (6) reported that serum specimens from man, horse, cow, dog, cat, rabbit, and guinea pig act as inhibitors of bacillary growth. In the present work, the effects of various mammalian serum samples upon the growth of tubercle bacilli were tested and an investigation made of ways of purification of the tuberculostatic factor which is present in most of the tested serum.

EFFECT OF TWEEN 80 ON THE GROWTH OF TUBERCLE BACILLI IN AERATED CULTURES.

Lyon, Richard H. (Veterans Administration Hospital, Minneapolis, Minn.), Herman C. Lichstein, and Wendell H. Hall. Effect of Tween 80 on the growth of tubercle bacilli in aerated cultures. J. Bacteriol. 86:280-284. 1963.-The effect of Tween 80 (polyoxyethylene sorbitan monooleate), glucose, and glycerol on aerated and stationary growth of Mycobacterium tuberculosis strain H(37)Ra was examined in Dubos liquid medium. Previous studies established that aeration (rotation) of liquid cultures of M. tuberculosis strains H(37)Ra and H(37)Rv caused an inhibition of growth in a medium containing glucose as a source of carbohydrate. The present studies show that Tween 80 exerts a toxic effect on the growth of tubercle bacilli in aerated cultures when glucose is present in the medium as the sole source of carbohydrate, but not when glycerol is included. The role of hydrolytic products of Tween 80, viz. oleic acid and the polyoxyethylene derivative of sorbitol, is discussed. The hypothesis is submitted that glycerol protects against the growth suppression by aeration because it reduces the concentration of free fatty acids in the medium to subinhibitory levels.

Slide culture of tubercle bacilli. IV. Further diagnostic trials.

THE CONSTANTLY increasing number of publications concerning media and methods designed to provide more accurate, more rapid, and more reliable laboratory diagnosis of tuberculosis attests to the fact that at present no one method is entirely satisfactory. One of the problems has been to devise a procedure which would permit an earlier bacteriological diagnosis of the disease and which would be of greater assistance in clinical management during its course. It has been pointed out by Cummings' that the ideal culture medium should support rapid growth of small numbers of tubercle bacilli, should permit easy differentiation by colonial morphology between pathogenic and nonpathogenic acid-fast bacilli, should inhibit growth of contaminants, and should be simply and reproducibly prepared from readily available ingredients. Most of the media used in the diagnostic isolation of tubercle bacilli today do not satisfy all of these requirements. They contain complex organic materials such as animal sera, egg yolk, or potato extract, none of which can be reproducible from one batch to another. During the past ten years, however, a number of media which meet most or all of the requirements of the ideal culture medium have been devised. The development of these media and methods has been reviewed recently by Reed and Morgante.2 One of these is the slide microculture method originally described by Koch3 by which he was able to demonstrate growth of tubercle bacilli within a week. Adaptations of this method by Pryce4 and Rosenberg 5 for diagnostic use were very sensitive, but too cumbersome for routine use. A notable advance in technic was reported by Lowry and Berry 6 with the development of special culture tubes and decontamination tubes which permitted simple and safe handling of sputum smears dried on ordinary slides. Reed 7 in 1953 reported a modification of the slide culture method which yielded excellent results, and subsequently Simpson and Reed 8 carried out fairly extensive trials of the modified technic in a routine diagnostic laboratory. It was found that slide culture appeared to be superior to conventional methods particularly in terms of the number of positive cultures produced and in the time required for them to become positive. These diagnostic trials have been carried on for the past three years. It has always been accepted as precept that the use of a variety of media should produce an appreciable increase