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OBJECTIVES: The aim of this study was to determine the antimycobacterial potential of laurel oil, its fractions and its two sesquiterpene lactones against several mycobacterial strains and clinical isolates, and to establish the possibility of occurrence of some synergistic effects between those lactones using a modification of the fluorometric Alamar Blue microassay (FMABA). METHODS: The in vitro antimycobacterial activity of whole oil and its fractions and pure active compounds were determined by FMABA. A bioassay-guided fractionation of the traditional preparation of laurel oil from Madeira Islands was performed, yielding pure compounds chemically identified by standard procedures. Synergism of pure compounds was established by X/Y quotient analysis adapted to FMABA. RESULTS: Sesquiterpene lactones, costunolide and dehydrocostuslactone, were the compounds responsible for the antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MICs of 6.25 and 12.5 mg/L, respectively. Antimycobacterial activity against drug-resistant M. tuberculosis clinical isolates was better for the mixture than for pure compounds. CONCLUSIONS: Both lactones presented synergistic activity, i.e. analysis of relative fluorescence units presented an X/Y value <0.5 at a concentration of 1/8 MIC of each compound in the combination. Establishment of synergism by FMABA represents another application of the microplate Alamar Blue assay.

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The aim of this study was to apply receiver operating characteristic (ROC) analysis to the microplate Alamar blue assay, a recently developed alternative for drug susceptibility testing of mycobacteria. As this is a quantitative assay, its performance can be determined by ROC analysis, in which the area under the ROC curve represents a summary of test performance (the higher the area, the better the test's performance). Sixty isolates of Mycobacterium tuberculosis were tested by the microcolorimetric assay against six twofold dilutions of streptomycin, isoniazid, rifampin, and ethambutol. For each isolate, the susceptibility pattern was simultaneously established by the agar proportion method, the result of which represented the gold standard value for the ROC analysis. The critical concentration, area under the curve, and P value for each drug were determined by ROC curve analysis. The results of the assay were obtained in an average of 8 days of incubation. The performance of the assay was excellent for all four drugs: the area under the curves was >0.97, the P values were 0.000, and sensitivity was 94%, specificity 97%, predictive value for resistance >/=92%, predictive value for susceptibility 97%, and test efficiency 97%. According to ROC analysis, the microplate Alamar blue assay is a reliable method for determination of drug-susceptibility. Rapidity and cost efficiency are two additional qualities that make this test an excellent alternative for the drug susceptibility testing of Mycobacterium tuberculosis. The ROC curve analysis is a robust statistical approach for evaluating the performance of new quantitative methods for determination of drug sensitivity of Mycobacterium tuberculosis isolates.

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SETTING: Differential diagnosis of leprosy and tuberculosis in regions where both illnesses are endemic is a prerequisite for proper identification and treatment. OBJECTIVE: To evaluate the recognition of phenolic glycolipid-I (PGL-I) of Mycobacterium leprae and sulfolipid-I (SL-I) of M. tuberculosis by serum from patients with leprosy (LL) or pulmonary tuberculosis (PTB). DESIGN: Purified PGL-I and SL-I were used as antigens in an ELISA test set up to assess recognition of these lipids by serum from 43 LL patients, 44 PTB patients and 38 healthy individuals. RESULTS: Leprosy patients gave higher IgM than IgG responses to PGL-I and had comparable IgM and IgG responses to SL-I. A similar situation was observed with PTB serum. Some healthy individuals were found to contain significant levels of antibodies to both lipids. CONCLUSION: There is no specific recognition of either of the two lipid antigens tested by serum from both leprosy and tuberculosis patients; this rules out the possibility of using PGL-I and SL-I as tools for the differential diagnosis of these two mycobacterial diseases.

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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.

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Lipids extracted from mouse tissues infected with Mycobacterium lepraemurium (MLM) were analyzed by thin-layer chromatography. Although the extracted lipids were heterogeneous in polarity, the lipids of intermediate polarity were the ones that predominated. All of the lipids of intermediate polarity were glycosylated species. There were also lipids of low and high polarity, the latter being glycolipids. Compared to lipids extracted from normal tissue (mostly to lipids of high and low polarity), all of the additional lipids extracted from the infected tissue corresponded to lipids present in the purified bacteria. Enzyme-linked immunoassays (ELISAs) were then performed with the whole lipids extracted from purified bacilli, the lipids of high, intermediate and low polarity, and the sera from 20 normal and 20 MLM-infected mice. Lipids of intermediate polarity were specifically recognized by MLM-infected mice. Neither sera (diluted 1:500) from normal mice nor infected mice reacted with the lipids of high or low polarity, but a higher concentration (sera diluted 1:100) of some sera from mice in both groups reacted significantly with these lipids. In the ELISAs the whole-lipid extract and the lipids of intermediate polarity were similarly recognized by the sera of the infected mice. Thus, as observed in human leprosy, the mycobacterial disease in the mouse (murine leprosy) is also accompanied by the development of antibodies to the glycolipids of the infecting microorganism.

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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.

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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.

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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.

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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.

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We investigated the activity of benzoxazinorifamycin (KRM-1648) against several drug-susceptible and multidrug-resistant strains of tubercle bacilli. Since KRM-1648 is a rifamycin derivative, we included some strains of Mycobacterium tuberculosis resistant to rifampin (RIF) among the multidrug-resistant strains. For RIF-susceptible strains, the MIC of KRM-1648 was much lower than that of RIF (MICs of KRM-1648 and RIF at which 90% of strains are inhibited, < or = 0.015 and < or = 0.25 micrograms/ml, respectively). The MBC of KRM-1648 (range, 0.007 to 0.03 microgram/ml) was also much lower than that of RIF (range, 0.5 to 1.0 microgram/ml). Postantibiotic effect studies with KRM-1648 showed a rapid reduction in the CFU counts with an exposure of 24 h or more, and its sterilizing effect was maintained even up to 21 days thereafter. Parallel postantibiotic effect studies with RIF showed a less significant effect with a faster recovery of growth, and RIF failed to sterilize the organisms even after 72 h of exposure. KRM-1648 at 0.125 and 0.25 microgram/ml caused complete inhibition of intracellular growth of M. tuberculosis in J774 A.1 macrophages after 48 h of exposure. After a similar exposure time RIF at a concentration of 0.25 microgram/ml caused complete inhibition of growth, but a concentration of 0.125 microgram/ml caused only a 50% reduction in growth compared with that of controls at day 7. With 24 h of pulsed exposure of the intracellular organisms to 0.25 micrograms of the drugs per ml, KRM-1648 caused complete inhibition of intracellular growth, while RIF caused only moderate inhibition of intracellular growth. These findings suggest that KRM-1648 is a potentially useful drug for the treatment of tuberculosis.

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Mycobacterium avium complex (MAC) strains from AIDS and non-AIDS patients and from the environment were studied for their colony morphology and virulence in beige mice. The majority of the MAC isolates from AIDS patients, in contrast to those from non-AIDS patients and the environment, showed increased virulence. Similarly, the majority of the MAC isolates from AIDS patients formed smooth transparent (ST) colonies, whereas most of the non-AIDS isolates formed smooth opaque (SO) or intermediate (IM) type of colonies. MAC isolates from the same AIDS patient obtained at different times were found to be heterogenic with respect to serotype, RFLP and glycolipid patterns, suggesting that these patients might be infected with more than one strain of MAC.

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