Comparison of several culture media used for studies on mycobacteriophages.

The value of RVA, N-1, 7H10, 7H11 and Sauton's media for studies on mycobacteriophage infeciton and lysis of mycobacteria was assessed. Experiments were made with mycobacteriophages BGI, BKI, CRI-3, G37, and LG, all of which lyse Mycobacterium smegmatis strain 607B, and with mycobacteriophage DS6A which lyses Mycobacterium tuberculosis strain H37Rv. The methods involved "direct lysis", the measurement of "routine test dilutions" and counts of plaque-forming units. It was found that N-1, 7H10 and 7H11 media gave better overall results than RVA medium for M. smegmatis strain 607B and its phages, and that RVA medium was generally the most useful for M. tuberculositems employed.

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.

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.

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.

Utility of beige mouse in leprosy research.

Dissemination of M. leprae to visceral organs is seen by four months onwards only in beige (C57BL/6/bgj) but not BALB/c mice following intravenous or intraperitoneal infections. Inoculation of the beige mouse derived M. leprae showed all the characteristics of M. leprae, including growth pattern in the foot-pads of BALB/c mice. M. leprae inoculated into foot-pads of beige mice multiplied faster than those in the foot-pads of BALB/c mice. The possibility of using beige mouse in chemotherapeutic studies in leprosy is discussed.

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.

Murine models for mycobacterioses.

Murine models have proven very useful in studying the pathobiology and chemotherapy of a number of mycobacterioses. While there are a number of significant variations from the human model, a substantial body of experience aids in extrapolating from the murine model to the human condition. The most extensively studied infection is Mycobacterium tuberculosis. However, there have been significant advances recently using the Beige mouse (an immunodeficient mutant) as an acute infection model for disease due to Mycoba avium complex. Less extensively studied have been Mycobacterium kansasii, Mycobacterium fortuitum, and Mycobacterium chelonei, the other relatively common nontuberculous mycobacterioses involving man.

In vivo depletion of natural killer cell activity leads to enhanced multiplication of Mycobacterium avium complex in mice.

Earlier investigations have shown that murine natural killer (NK) cells inhibit the growth of fungal and bacterial pathogens in vivo and in vitro. In order to define the role of NK cells in Mycobacterium avium complex infection, in vivo depletion of NK cells by using anti-NK1.1 monoclonal antibody and conventional anti-asialo-GM1 antibody has been attempted. Repeated injection of 200 micrograms of anti-NK1.1 or 50 micrograms of anti-asialo-GM1 antibody effectively depleted NK activity in the spleens of C57BL/6 mice. The growth kinetics of M. avium complex over a period of 4 weeks showed that the colony counts in the spleens of the antibody-treated group were significantly (P less than 0.01) higher than those of the control group and compared well with those of the genetically NK cell-deficient C57BL/6 bg/bg mutant. The alternate strategy of in vivo stimulation of NK activity by poly(I:C) administration did not show a similar reduction in CFU in the spleen compared with the untreated control. The in vivo antibody depletion of NK activity provides direct evidence on the role of NK cells in the control of intracellular mycobacterial pathogens such as M. avium complex.

In-vivo activity of streptomycin and clofazimine against established infections of Mycobacterium avium complex in beige mice.

Beige mice were challenged with 10(6)-10(7) cfu of Mycobacterium avium intracellulare strain 101 and 22 days later treated with streptomycin 150 mg/kg/day alone, clofazimine 20 mg/kg/day alone, streptomycin 150 mg/kg/day plus clofazimine 20 mg/kg/day, or no antimicrobial agent (untreated controls). Both single-drug therapies partially reduced the cfu counts in spleen, liver and lungs compared with the controls however the combination was significantly more effective and completely eliminated the pathogen from the spleen and lungs of some animals after eight weeks treatment.

Heat shock treatment of macrophages causes increased release of superoxide anion.

Heat shock treatment of murine macrophages and the J774 cell line resulted in an enhanced capacity to release superoxide anion (O2-) upon stimulation. There was no concomitant increase in hydrogen peroxide production, and the macrophage microbicidal activity against Mycobacterium tuberculosis, Mycobacterium avium complex, and Staphylococcus aureus was not altered.

In vitro response of murine alveolar and peritoneal macrophages to Mycobacterium intracellulare.

Normal resident and BCG-activated alveolar and peritoneal macrophages from female Swiss Webster mice were compared for their ability to ingest and subsequently control the multiplication of Mycobacterium intracellulare in vitro. Resident peritoneal macrophages failed from the moment of ingestion to control the multiplication of engulfed bacilli resulting in host cell lysis, whereas activated peritoneal macrophages and both resident and activated alveolar macrophages constrained bacterial division for at least 7 days before comparable bacterial multiplication led to phagocyte death. The number of bacilli needed to lyse a macrophage was impossible to determine precisely because viable macrophages commonly contained several hundred mycobacteria. Minimal intracellular bacterial generation times were 20 h for each macrophage type. Differences in the rates of bacterial phagocytosis between both macrophage types, either resident or activated, are intrinsic properties of the macrophages and were not induced by the mycobacteria, because the same patterns of particle ingestion were observed after exposure to latex microspheres.

Release of superoxide anion from resident and activated mouse peritoneal macrophages infected with Mycobacterium intracellulare.

Phagocytosis of Mycobacterium intracellulare triggered the release of superoxide anion (O-2) from mouse peritoneal macrophages; the amount release from BCG-activated cells was significantly greater than that from resident cells. Superoxide anion release was proportional to the number of phagocytes ingesting bacilli. An optimal phagocytic time course of 4 h was observed when estimating O-2 release consequent to bacterial challenge. Also, an inverse relationship between the amount of O-2 released and the virulence of M. intracellulare in mice was shown. Transparent and rough colony variants triggered the release of significantly lower amounts of O-2 as compared with the opaque and smooth colony variants. Serovars 4 and 8, which cause a disease with a poor prognosis in humans, triggered significantly less O-2 than did serovars 9, 12, 13, 14, 16, 18, and 19, which respond more favorably to chemotherapeutic regimens.