In vitro anti-mycobacterial activities of three species of Cola plant extracts (Sterculiaceae).

Extracts obtained from three Nigerian Sterculiaceae plants: Cola accuminata, C. nitida and C. milleni were screened for anti-mycobacterium properties using a slow growing Mycobacterium bovis ATCC 35738 (designated BCG Mexican and known to have some virulence in mouse and guinea pig) at 1000 microg/ml using the radiometric (BACTEC) method. The extracts were also tested against six fast growing ATCC strains of M. vaccae using the broth microdilution method. The methanol extracts from both leaves, stem bark and root bark of Cola accuminata and from the leaves and stem bark of C. nitida and C. milleni were not active at the highest concentration of 1000 microg/ml. Only the methanol extract of root bark for both C. nitida and C. milleni were found to be potent against both M. bovis and strains of M. vaccae. The minimum inhibitory concentration (MIC) of C. nitida against M. bovis is 125 microg/ml while the MIC of C. milleni against M. bovis is 62.5 microg/ml after at least 6 days of inhibition with growth index (GI) units lesser than or equal to the change in GI units inoculated with a 1/100 of the BACTEC inoculum for a control vial. The minimum inhibitory concentration of C. milleni against the six ATCC strain of M. vaccae ranged from 62.5 microg/ml to 250 microg/ml while for C. nitida ranged from 500 microg/ml to above 1000microg/ml. Evidently, C. milleni has the highest inhibitory activity against both M. bovis and strains of M. vaccae used. Rifampicin, the positive control used has strong activity against M. bovis at the tested concentration of 5 microg and 10 microg/ml and 4 to 8 microg/ml against the six strains of M. vaccae.

Direct drug susceptibility test for tubercle bacilli by the sputum swab culture method.

OBJECTIVE: To develop a simple, inexpensive method for testing direct drug susceptibility of tubercle bacilli to isoniazid (INH) and streptomycin (SM) which can be adopted for use even in remote parts of the world. DESIGN: Using 239 smear-positive sputum specimens obtained from an equal number of patients, a comparison was made between the direct swab susceptibility test and the standard indirect method for INH and SM using Löwenstein-Jensen (L-J) medium. RESULTS: There was 95% agreement of results for INH by 6 weeks and 90% for SM by 8 weeks; 96% of INH-resistant cultures could be detected in 5 weeks and 91% of SM-resistant cultures by 8 weeks. The discrepancies in the two tests were virtually symmetrically distributed at 6 and 8 weeks. The speed and efficiency of detection of resistance by the swab method was also assessed in relation to the standard indirect method. For INH, 96% of the cultures were detected by the fifth week, while 66% were detected as early as 2 weeks and 93% by 4 weeks. With SM, 84% were detected by 4 weeks, 89% by 5 weeks and 91% by 8 weeks. CONCLUSION: This study has indicated the usefulness of the swab method for testing the drug susceptibility of tubercle bacilli. As this method is simple and easy, and does not even require a centrifuge, it has the potential of application even in the remote parts of the world. The material used, Cetavlon (Cetrimide), is inexpensive and easily water soluble, and more importantly, aqueous solutions are self-sterilizing. It should be stressed, however, that the results obtained with this test take the same time as conventional methods, and it can therefore not be considered as a rapid test.

Antimycobacterial activities of riminophenazines.

Riminophenazines were specifically developed as drugs active against Mycobacterium tuberculosis but extensive research over several decades has shown that these compounds are also active against many other mycobacterial infections, particularly those caused by Mycobacterium leprae and the Mycobacterium avium complex (MAC). Clofazimine, the lead compound in this series, is included in the regimens that are approved by the WHO for the treatment of leprosy and has contributed significantly to the control of that disease, particularly that caused by dapsone-resistant bacteria. Despite early problems, clofazimine has shown clinical efficacy in tuberculosis, in particular that caused by multiple drug resistant strains. Clofazimine does not induce resistance and also inhibits emergence of resistance to isoniazid in M. tuberculosis. The efficacy of clofazimine against MAC is more varied and the availability of better drugs has limited its use. Newer riminophenazines, such as B746 and B4157, not only showed increased anti-mycobacterial activity but also produced less skin pigmentation, which is the main drawback of this group of compounds. The most important virtues of riminophenazines, such as intracellular accumulation in mononuclear phagocytic cells, anti-inflammatory activity, a low incidence of drug resistance and slow metabolic elimination, make them attractive candidates for the treatment of mycobacterial infections. It is essential, however, to investigate the newer analogues clinically, while continuing the pursuit of alternate candidates that demonstrate higher anti-mycobacterial activity and lower rates of skin pigmentation.

Chemotherapy of tuberculosis in mice using single implants of isoniazid and pyrazinamide.

OBJECTIVE: To establish the chemotherapeutic value of a depot drug preparation of isoniazid and pyrazinamide against experimental tuberculosis. DESIGN: To see whether sustained levels of pyrazinamide are available for prolonged periods after a single subcutaneous administration of a biodegradable polylactic-glycolic acid (PLGA) polymer containing the drug, studies were done to ascertain whether a single administration of isoniazid and pyrazinamide in separate PLGA polymers could offer chemotherapeutic protection against a heavy intravenous challenge of susceptible mice with a virulent strain of Mycobacterium tuberculosis similar to that rendered by daily administration of the two drugs for 8 weeks. RESULTS: Even with three times the daily dose of pyrazinamide contained in the single PLGA polymer implant, no abnormally high (burst) levels of the drug were evident after administration, but sustained levels of the drug were seen up to 54 days. The chemotherapeutic activity of the single PLGA polymer implants was similar to that obtained with standard oral treatment with the two drugs given daily for the entire 8 weeks, as judged by mortality and colony forming unit (CFU) counts of tubercle bacilli from lungs and spleen. CONCLUSION: Treatment with single implants of the PLGA polymer containing anti-mycobacterial drugs offers a strong possibility of circumventing the compliance problem.

Contribution of rpoB mutations to development of rifamycin cross-resistance in Mycobacterium tuberculosis.

The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the beta-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutant rpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoB mutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specific rpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.

Effect of polymer foam morphology and density on kinetics of in vitro controlled release of isoniazid from compressed foam matrices.

The purpose of this study was to compare the effect of polymer foam morphology and density prior to compaction on the kinetics of isoniazid (INH) release from the final high-density extruded matrices. The feasibility of preparing low density foams of several biopolymers, including poly(L-lactide) (PLLA), poly(glycolide) (PGA), poly(DL-lactide-co-glycolide) (PLGA), poly(gamma-benzyl-L-glutamate) (PBLG), and poly(propylene fumarate) (PPF), via a lyophilization technique was investigated. Low-density foams of PLGA, PBLG, and a mixture of PLGA and PPF were successfully fabricated by lyophilization of the frozen polymer solutions either in glacial acetic acid or in benzene. The morphology of these foams depends on the polymer as well as the solvent used in the fabrication process. Thus, PLGA produces a capillary structure when lyophilized from benzene solution and a leaflet structure from glacial acetic acid, but PBLG yields a leaflet structure from benzene. Matrices were prepared by impregnating these foams with aqueous solutions of INH, removing the water by a second lyophilization, and then compressing the low-density INH containing foams by compaction and high-pressure extrusion. The resulting nonporous matrices had densities of approximately 1.30 g/cm3. In vitro kinetics were in accord with the Roseman-Higuchi diffusion model and demonstrate that release rates depend on the initial foam density, while foam structure has little influence on the release kinetics.

Microbiology of nontuberculosis mycobacteria.

Recognition of differences, particularly in color and other growth characteristics, between Mycobacterium tuberculosis and other related organisms has led to the identification of the nontuberculosis mycobacteria (NTM). The Runyon Classification, based largely on the presence (or absence) of pigment and rate of growth, was a convenient system for classifying these organisms. Subsequent biochemical analysis has facilitated more precise speciation. However, many of these tests are complex and time consuming and beyond the capacity of most laboratories other than reference centers. The need for accurate identification has become clear because of differences in pathogenicity and treatment between various species and even subspecies of NTM. The recent appearance of rapid diagnostic tests and highly specific genetic identification systems have created new opportunities and challenges with respect to the identification of NTM. This article provides a historic context for the microbiologic study of the organisms, describes key features of the most commonly encountered NTM, notes several important new technical innovations in this field, and considers common clinical issues in relation to the microbiology of NTM.

Treatment of intracellular Mycobacterium avium complex infection by free and liposome-encapsulated sparfloxacin.

Mycobacterium avium-M. intracellulare complex (MAC) is the most frequent cause of opportunistic bacterial infection in patients with AIDS. Previous studies have indicated that liposome-encapsulated aminoglycosides are highly effective in treating MAC infections in mice. We investigated whether the fluoroquinolone sparfloxacin is effective in treating MAC infection in the murine macrophage-like cell line J774. Sparfloxacin was encapsulated in the membrane phase of multilamellar liposomes composed of phosphatidylglycerol-phosphatidylcholine-cholesterol (1:1:1 molar ratio). MAC-infected macrophages were treated for either 24 h or 4 days with free or liposome-encapsulated sparfloxacin. Treatment with free or liposome-encapsulated sparfloxacin (6 micrograms/ml) for 24 h resulted in the reduction of the growth index to 25 and 30% of that of untreated controls, respectively. When cultures were treated for 4 days, free sparfloxacin reduced the growth index to 6% of that of the untreated control, while liposome-encapsulated sparfloxacin reduced it to 8% of that of the control.

Pathobiological significance of colony morphology in Mycobacterium avium complex.

Mycobacterium avium complex (MAC) strains are known to exhibit variation in colony morphology. In addition to the smooth transparent (ST), smooth opaque (SO) and rough opaque (RO), which are the most common morphological forms, intermediate (IM) and pin point (PP) forms were also occasionally observed. In order to understand the pathobiological significance of these different colony forms, we investigated their virulence in beige mice, ability to bind to plastic and epithelial cells, differences in the lipids, and modulation of macrophage functions by the bacillary extracts. ST variants, the most common form seen in AIDS patients, were more virulent with increased multiplication in lungs, livers and spleens of beige mice and showed increased adherence to plastic and epithelial cells. SO, RO, PP colonial forms did not show increase in growth in any of the organs over a period of 4 weeks. IM colonial variants showed increased growth in lungs and spleens but not in livers. Thin layer chromatographic (TLC) analysis of lipid extracts showed one specific component in the high polar lipids of the SO variant, while ST variant did not show any specific component in any of the three families of lipids (high, intermediate and low polarity). The RO variant either expressed low levels or lost many of the components of lipids of high and intermediate polarity, however produced increased levels of lipids of low polarity. One of the components of low polar lipids was specific for RO variant and was produced in large quantity. The isogenic variants differed in the total lipid and sugar contents and also differed in their ability to modulate macrophage functions.

Mechanisms of isoniazid release from poly(d,l-lactide-co-glycolide) matrices prepared by dry-mixing and low density polymeric foam methods.

The release mechanisms of a small molecular drug from biodegradable poly(d,l-lactide-co-glycolide) (PLGA) cylindrical matrices were investigated. Isoniazid (INH), one of the most effective drugs against tuberculosis (TB), was selected as the model drug. Controlled-release matrices consisting of the drug and polymer were fabricated by two methods. The first of these, the dry-mixing method, involved the extrusion of a mixture of micronized drug and polymer particles as rods. In the second technique, the low density polymeric foam method, drug particles were enclosed in the cells of porous polymeric foams prior to extrusion. In vitro, the dry-mixed matrices released INH more rapidly than the polymeric foam matrices. The Roseman-Higuchi diffusion model, which had previously been found to be effective in analyzing the release kinetics of INH from the dry-mixed matrices, also fit the kinetics of INH released from matrices prepared from polymeric foams. This indicated that the release was still diffusion-controlled rather than degradation-controlled. The release mechanisms were further investigated, and two diffusion mechanisms, pore diffusion and lattice diffusion, were proposed for the INH controlled-release matrices according to the way in which they were prepared. Matrices prepared by the dry-mixing method appear to segregate drug particles along polymer grain boundaries and thus have a pore diffusion mechanism, while matrices prepared by the foam method entrap drug within the porous structure of foams and thus display a lattice diffusion mechanism. Theoretically, these two diffusion mechanisms can be identified by their activation energies for diffusion. With varying in vitro temperature, the activation energies were calculated from plots of ln (DIT) vs T-1 and in D vs T-1, where D is the diffusivity and T is the in vitro temperature in K. According to the results, we concluded that the INH from the dry-mixed matrices diffused through the drug channels filled with the medium, while the INH from the foam matrices diffused through the polymer lattice.

Chemotherapeutic activity of benzoxazinorifamycin, KRM-1648, against Mycobacterium tuberculosis in C57BL/6 mice.

OBJECTIVE: To investigate the chemotherapeutic activity of benzoxazinorifamycin, KRM-1648, in comparison with rifabutin (RFB) and rifampin (RIF) against experimental tuberculosis. DESIGN: C57BL/6 mice were infected with 10(5)-10(6) colony forming units (CFU) of either drug-susceptible virulent Mycobacterium tuberculosis (H37Rv) or multi-drug resistant (MDR) M. tuberculosis strain (2230) and were treated from the next day (early treatment) or after 2 weeks following infection (established infection) with 20 mg/kg dose of each drug or none (untreated control). The efficacy of chemotherapy was assessed based on prevention of mortality and on CFU levels in the lungs and spleens. RESULTS: All three drugs prevented mortality for up to 28 weeks of observation, while all the untreated control mice died by 4 weeks. Analysis of CFUs revealed superior therapeutic activity of both KRM-1648 and RFB as compared to RIF against the drug-susceptible strain of M. tuberculosis under the early treatment protocol. Twelve weeks' treatment with KRM-1648 or RFB caused complete sterilization of the lungs. However, residual organisms started appearing in the spleens 6 weeks after cessation of treatment with RFB and 16 weeks after KRM-1648 treatment. In mice infected with a MDR strain of M. tuberculosis, which was susceptible in vitro to KRM-1648, the drug did not appear to have any activity. Since the MDR organisms did not multiply in vivo, and did not cause any mortality up to 28 weeks in the RIF-treated control mice, a state of semi-dormancy of the organisms which might prevail in vivo could be responsible for refractoriness to treatment with KRM-1648. CONCLUSIONS: KRM-1648 showed an excellent chemotherapeutic activity, as compared to RFB and RIF, against drug-susceptible tuberculosis. However, all three analogues were ineffective against infection with multi-drug resistant strain of M. tuberculosis.

Polymerase chain reaction detection of Mycobacterium tuberculosis in formalin-fixed tissue.

Use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissue would be of great diagnostic value. However, formaldehyde has been reported to decrease the efficiency of amplification by structurally altering the polynucleotide chain. We sought to determine the ability of the PCR assay to detect M. tuberculosis in formalin-fixed, paraffin-embedded tissue in a mouse experimentally infected with the H37Rv strain of M. tuberculosis. Lung tissue from the infected mouse was cultured to determine the number of organisms per gram of tissue. The remaining lung tissue was divided into eight portions, seven of which were fixed in 10% neutral buffered formalin for 24-h intervals over periods lasting from 1 to 7 d, and a control portion was placed in isotonic saline. The tissue samples were then paraffin-embedded, and sections were obtained from each tissue block for PCR analysis. We show that the PCR assay can detect as few as nine organisms in a 5-micron section of tissue, and that up to 7 d of fixation in 10% neutral buffered formalin has a negligible effect on the assay. The PCR assay can detect low numbers of M. tuberculosis organisms in formalin-fixed, paraffin-embedded tissue.

Antituberculosis activities of clofazimine and its new analogs B4154 and B4157.

In our efforts to develop new drugs for the treatment of tuberculosis, especially that caused by multidrug-resistant strains, we investigated clofazimine (CFM) and two of its analogs, B4154 and B4157, for their antituberculosis activities. Twenty M. tuberculosis strains were tested, including 16 drug-resistant strains (strains resistant to one or more antituberculosis drugs), for their susceptibilities to these three agents. All of the strains were found to be susceptible to B4154 and B4157, and one strain showed moderate resistance to CFM. The MICs of B4154, B4157, and CFM at which 90% of strains were inhibited were 0.25, 0.12, and < or = 1.0 microgram/ml, respectively. The intracellular activities of CFM and B4157 were superior to that of B4154. The chemotherapeutic activities of the three compounds were evaluated in C57BL/6 mice. At a dose of 20 mg/kg of body weight, the activity of CFM was slightly superior to that of B4157; however, both compounds prevented mortality and caused a significant reduction in the numbers of CFU in the lungs and spleens. The animals treated with B4157 showed less pigmentation than animals treated with CFM. The chemotherapeutic activity of CFM was comparable to those of rifampin and isoniazid. Complete susceptibility of multidrug-resistant strains to CFM and B4157 and the therapeutic efficacies of these compounds against mouse tuberculosis make these drugs attractive agents for the treatment of drug-resistant tuberculosis.

Antituberculosis activity of clarithromycin.

Antituberculosis activity of clarithromycin (CLA), a macrolide antibiotic, was investigated in vitro, in macrophages, and in C57BL/6 mice, CLA showed high in vitro MICs (4 to > 16 micrograms/ml) for several strains of Mycobacterium tuberculosis and caused slight enhancement of activity of rifampin (RIF) against H37Rv but failed to increase the activity of either RIF or isoniazid (INH) against other strains. However, inside J774A.1 macrophages, CLA showed high activity and was synergistic with RIF against some strains of tubercle bacilli susceptible or resistant to INH and RIF. In the in vivo studies with a drug-susceptible strain (H37Rv), CLA protected mice from mortality due to tuberculosis for up to 8 weeks of observation. The CFU data for lungs and spleens revealed that the antituberculosis activity of CLA is inferior to those of INH and streptomycin. However, the activity of CLA when used alone or in combination was comparable to that of thiacetazone, indicating its potential usefulness as a secondary drug for the treatment of tuberculosis.

Antimycobacterial activity of a new rifamycin derivative, 3-(4-cinnamylpiperazinyl iminomethyl) rifamycin SV (T9).

The antimycobacterial activities of a new rifampin (RIF) derivative, 3-(4-cinnamylpiperazinyl iminomethyl) rifamycin SV (To), against 20 susceptible and multidrug-resistant strains of Mycobacterium tuberculosis and 20 Mycobacterium avium complex (MAC) strains were investigated. The radiometric MICs of T9 for M. tuberculosis were significantly lower than those of RIF. The MICs of T9 and RIF at which 90% of the RIF-susceptible strains were inhibited were < or = 0.25 and < or = 0.5 micrograms/ml, respectively. Interestingly, T9 had lower MICs against some RIF-resistant M. tuberculosis strains. T9 had better activity against MAC strains, and the MIC at which 90% of the MAC strains were inhibited was < or = 0.125 micrograms/ml, and that of RIF was < or = 2.0 micrograms/ml. T9 also showed high in vitro bactericidal and intracellular activities which were significantly superior to those of RIF against both M. tuberculosis, and MAC strains. More importantly, T9 showed excellent in vivo activity against M. tuberculosis H37Rv compared with that of RIF in both the lungs and spleens of C57BL/6 mice, indicating the potential therapeutic value of T9 in the treatment of mycobacterial infections.

In-vitro and intracellular activity of rifabutin on drug-susceptible and multiple drug-resistant (MDR) tubercle bacilli.

Rifabutin, a spiropiperidyl derivative of rifampicin, is approved for the prophylaxis of Mycobacterium avium infections in AIDS patients in the US, and for the treatment of M. avium infections, tuberculosis and multiple drug resistant tuberculosis in many countries. In the present study, rifabutin was compared with rifampicin for its activity against drug susceptible and multi-drug resistant tubercle bacilli by several in-vitro and macrophage studies. Rifabutin exhibited similar or greater in-vitro activity than rifampicin as judged by the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and MBC/MIC ratios, as well as continuous exposure and post-antibiotic effect studies. Rifabutin has been shown to be active against some multiple drug resistant strains which were resistant to rifampicin. In macrophage studies with continuous exposure to the drug or when the drug had been removed after 24 h, rifabutin also demonstrated high activity which was better than RMP against intracellular tubercle bacilli. This long-acting intracellular anti-mycobacterial activity may explain, in part, the clinical efficacy of rifabutin.