No nosso país são actualmente utilizadas diversas tuberculinas administradas em doses variáveis, pelo que procedemos à avaliação do padrâo de reactividade à tuberculina RT23 (State Serum Institute, Copenhaga na população portuguesa. Usámos para essa investigação 2 unidades tuberculínicas (UT) de tuberculiua RT23.

The genes controlling the biosynthesis of the carotenes inMycobacterium aurum were clustered in a 10.83-kb segment. Fragments generated by endonuclease digestions of the segment were cloned into a pHLD69 shuttle vector. The plasmids so constructed were used to transform a colorless (albino)M. aurum mutant (strain A11), a brick-red mutant accumulating large amounts of lycopene (strain NgR9), the buff-coloredMycobacterium smegmatis MC2-155, and the buffcoloredMycobacterium tuberculosis H37Ra. From the endonuclease digestion patterns and the phenotypes of the transformed strains, the partial physical and functional maps of a carotenogenesis operon were established. This investigation also showed that the genes controlling the conversion of lycopene into the xanthophylls were not located in the 10.83-kb segment.

The genes controlling the biosynthesis of carotenes in responsible for its yellow pigmentation were previously cloned (FEMS Microbiol Lett 1992, 90:239–244). In this study, the genes α, and, controlling respectively the formation of lycopene from phytoene (phytoene desaturase), α-carotene from lycopene, and leprotene from lycopene, were localized.

The genes controlling the biosynthesis of carotenes inMycobacterium aurum responsible for its yellow pigmentation were previously cloned (FEMS Microbiol Lett 1992, 90:239–244). In this study, the genescrt I, car α, andcar Lep, controlling respectively the formation of lycopene from phytoene (phytoene desaturase), α-carotene from lycopene, and leprotene from lycopene, were localized.

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC2155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×105 forM. smegmatis MC2155, 6×103 forM. tuberculosis H37Ra, 103 forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.

J-774 murine macrophages were allowed to multiply in the presence of gammairradiated (4.5×106 rads)Mycobacterium avium for 3 days. The macrophages thus stimulated and still containing killed bacteria were then challenged with viableM. avium bacteria, and the intracellular growth of these bacilli was measured during 1 week by lysing the J-774 cells and measuring the viable bacterial counts on 7H10 ager. The results were compared with those obtained in parallel with normal J-774 cells. Our results showed that pretreatment of macrophages with killedM. avium neither enhanced their capacity to check the intracellular growth of viableM. avium nor did it potentiate the intracellular activity of rifampicin, ansamycin and clofazimine against actively multiplyingM. avium.

The intracellular growth kinetics ofMycobacterium xenopi was studied in the murine J-774 macrophage cell line model. During the initial 4 days of infection, the bacilli divided about every 33 h. Electron microscopy of infected macrophages showed that bacteria inside phagosomes were surrounded by a protective electron-transparent zone (ETZ). This model was used for comparing the extracellular and intracellular activities of the following drugs: pristinamycin (PRISTINA), isoniazid (INH), clofazimine (CLOFA), rifabutin (=ansamycin; ANSA), rifampicine (RIFA), streptomycin (SM), ethambutol (EMB), and five fluoroquinolones, namely, ciprofloxacin (CIPRO), ofloxacin (OFLO), pefloxacin (PEFLO), enoxacin (ENOX) and norfloxacin (NORFLO). All the drugs were tested within their obtainable serum level concentrations in man. Under these conditions, CLOFA, SM, CIPRO, and OFLO were highly active against intracellularly growingM. xenopi, INH and RIFA were moderately active, whereas ANSA, PRISTINA, EMB, PEFLO, ENOX, and NORFLO were only growth inhibiting. The comparison of these data with extracellular activities of the same drugs underlined the discrepancies observed in test-tube drug activity evaluation and its correlation with results of chemotherapy in patients in whom the drug has essentially an intracellular bacterial killing role.

Radiolabeled amino acids (l-U[C14]alanine,d-U[C14]alanine,l-U[C14]threonine, andl-U[C14]phenylalanine) were exponentially incorporated into the trichloroacetic acid (TCA)-insoluble material (whole cells) ofMycobacterium avium during the first 30–60 min of labeling. Bacteria labeled for 48 h were extracted with chloroform-methanol (2∶1 vol/vol). The thin layer chromatography (TLC) analysis of native lipids showed that mycoside C was labeled by the amino acids used.d-cycloserine (d-CS) and other amino acid analogs were examined as potential inhibitors of mycoside C biosynthesis. It was found thatd-CS caused about 27% inhibition, whereaso-,p-, andm-fluoro-dl-phenylalanine (Fl-phe) caused 80%–90% inhibition of the mycoside C biosynthesis. Judging from the data on inhibition experiments, it was concluded that the mycoside C biosynthesis started from the fatty acyl end and proceeded by the stepwise addition ofd-phenylalanine,d-allo-threonine, andd-alanine. Thed-alanyl-d-alanine peptidoglycan intermediate did not seem to serve as a donor ofd-alanine for mycoside C biosynthesis. Ultrastructural observation of the bacteria treated withd-CS showed only partial alteration of the outer wall layer, whereasm-Fl-phe treatment caused profound alterations. Successive transfers of the bacteria in growth medium supplemented withm-Fl-phe resulted in extensive disorganization of the outer layer.

In in vitro assays, the antibiotic pyrazinamide (PZA) is active against Mycobacterium tuberculosis in acidic medium (6), and intracellular activation of this drug in acidic loci of phagocytes (5) has long been proposed. However recent studies on PZA action on intracellular tubercle bacilli (2, 3) could establish only a bacteriostatic activity of the drug. Moreover, in one study PZA was added right from the beginning of phagocytosis (2), and consequently the bacilli could not undergo active multiplication before drug addition. Because we recently developed a J-774 murine macrophage cell line model for intracellular growth of mycobacteria (4), we decided to investigate the action of PZA on tubercle bacilli growing actively inside J-774 macrophages. M. tuberculosis H37Rv was grown in complete 7H9 medium (Difco Laboratories) containing 0.05% (vol/vol) Tween 80. Exponentially growing bacteria (optical density of 0.15 at 650 nm) were used to infect the macrophages for 4 h, and then their intracellular growth was established as reported earlier (4). Control experiments showed that bacilli did not grow significantly in the RPMI culture medium during the time alloted for these experiments (7 days). In one set of experiments, PZA (25, 50, or 100 ,ug/ml) was added after 48 h of intracellular growth of bacteria, and bacterial viability was measured after 2 and 5 days of drug addition on 7H10 agar medium (Difco), as reported earlier (4). In a second set of experiments, to avoid any eventual inhibition of drug penetration in already infected macrophages, we pretreated the macrophages with PZA (50 or 100 ,xg/ml) before phagocytosis. Since PZA penetration is almost complete within 3 h in mouse macrophages (1), we decided on a 4-h pretreatment. In this set of experiments, the drug was present throughout the course of infection and multiplication and was renewed at days 2 and 4.

The comparative action of seven drugs, namely, rifampicin (RIF), ansamycin-LM 427 (ANS), streptomycin (SM), isoniazid (INH), pyrizinamide (PZA), clofazimine (CLF), and pristinamycin (PST), was studied both on extracellularly and intracellularly growing mycobacterial speciesMycobacterium bovis, M. tuberculosis, andM. avium. All the drugs were used at their minimal inhibitory concentrations (MICs) and their obtainable serum levels in man; 10×MICs, when less than the obtainable serum levels, were also tested. The action of drugs on extracellularly growing bacilli was tested with the Middlebrook 7H9-Tween medium, whereas for intracellular growth, an experimental model using a continuous murine macrophage cell line J-774 was developed, and macrophage-mycobacteria interactions by use of cytochemistry, scanning and transmission electron microscopy were investigated. Extracellularly growingM. avium was resistant to all the drugs tested; however, when tested on intracellularly growing bacilli, both RIF and CLF were found to be bactericidal, whereas other drugs only delayed the bacterial growth. In the case of extracellularly growingM. bovis andM. tuberculosis, INH, RIF, and SM were active, whereas PZA was active only onM. tuberculosis. However, on intracellularly growing bacilli, only INH and RIF were found to be bactericidal for both species, CLF to a lesser extent in the case ofM. tuberculosis; ANS and SM were bacteriostatic, whereas both PZA and PST were without any antibacterial effect. This investigation underlined the discrepancies concerning the action of drugs on extra- and intracellularly multiplying mycobacteria and the advantage of using a continuous cell line model while studying drug susceptibility of mycobacteria in relation to their intracellular growth.

Treatment of mycobacterial cells with Triton X-100 allowed the extraction and solubilization of antigens. Such extracts provided species-specific crossed immunoelectrophoresis profiles which demonstrated that “Gordona aurantiaca” and Mycobacterium fallax are antigenically distinct. Using the Mycobacterium bovis BCG antiserum as a reference, we showed that M. fallax and M. bovis BCG are more related to each other than “G. aurantiaca” is to both species.

After phagocytosis by bone-marrow macrophages, Mycobacterium avium was surrounded by a thick electron-transparent zone (ETZ). The use of various fixation and embedding procedures showed that ETZ did not seem to be an artifactual structure. A quantitative assessment of ETZ frequency was performed at different times after infection of macrophages with SmD and SmT colony variants of M. avium. For SmT-variant-infected macrophages, a higher percentage of ETZ+ bacilli paralleled a higher percentage of intact bacilli than was the case for SmD-infected macrophages.

Ultrastructure of “Gordona aurantiaca”* M 296 (8128) was studied after the lead citrate coloration, whereas the cell envelope architecture was investigated by ruthenium red staining for outer wall acidic polysaccharides and the periodic-acid-thiocarbohydrazide-silver-proteinate cytochemical procedure (Thiéry method) for the detection of “α1-2 glycol bond containing polysaccharides.” The ultrastructural morphology of bacteria was distinct from both the mycobacteria and nocardia. The bacilli had a typical gram-positive cell wall that contained a thin, uniformly distributed, polysaccharide outer layer (POL) at its surface. The Thiéry cytochemical method stained only the cytoplasmic membrane, but not the cell wall, a feature that is common to the mycolic acid containing theCorynebacterium-Mycobacterium-Nocardia (CMN) group of organisms. The negative staining of the unfixed preparations of bacilli showed ribbonlike surface structures, common to the CMN group of organisms. The electron-microscopic preparations showed numerous lysing bacilli with bacteriophages indicating that the strain used was lysogenic.

An additional outer wall layer, composed mainly of acidic polysaccharides, was revealed in 18 species of mycobacteria by ruthenium red staining in electron microscopy. This report deals with the description of this additional layer. The ultrastructural implications at the level of the mycobacterial cell wall model and also the possible physiological role of this layer are briefly discussed.

We examined a small, 5.0-kb plasmid from Mycobacterium fortuitum, designated pAL5000. A restriction map of this plasmid was established. The complete sequence of pAL5000 was cloned in 3 different sites (BamHI, EcoRI, and EcoRV) of pBR322, and in the 3 possible orientations relative to the vector-derived sequence. The pBR322:: pAL5000 hybrids were used to transform the Escherichia coli mutant (minA, minB). Two of the hybrid plasmids (pAL15 and pAL51) coded for the same protein in the minicells, indicating that a sequence of mycobacterial plasmid DNA may function as a promotor recognised by the transcription-translation apparatus of E. coli.

Six plasmids differing in molecular weights were found in isolates ofMycobacterium fortuitum, M. chelonae, and other nonclassified, nonchromogenic, rapidly growing mycobacteria. One of the plasmids of molecular weight of 32.0 Kb was present in all strains analyzed; a large plasmid of 112.0-Kb molecular weight was present only in strains identified asM. fortuitum var.peregrinum. Although the strains had distinct plasmid profiles, none of the 15 cultural and biochemical properties and susceptibility to 18 drugs that were tested could be attributed to the occurrence of these plasmids.

Cell-envelope architectures of three strains of leprosy-derived corynebacteria (LDC) named Kim, FPSA, and 43LL were compared withMycobacterium leprae andM. avium as well as with related organisms (Nocardia asteroides andCorynebacterium psuedotuberculosis). Cytochemical studies were performed at the ultrastructural level after the lead citrate, silver proteinate, acidic phosphotungstic, and ruthenium red colorations. This study showed that, while the organisms belonging to theCorynebacterium-Mycobacterium-Nocardia (CMN) group had only the cytoplasmic membrane but not the cell wall reacting with the silver proteinate coloration, the LDC organisms had both the cell wall and the cytoplasmic membrane reacting with this coloration method. Moreover, the three strains of the LDC organisms differed from one another at the level of their exopolymer content.Mycobacterium leprae, on the other hand, gave a cytochemical response common to other mycobacteria and the members of the CMN group studied. Consequently, the envelopes of the LDC organisms were not identical toM. leprae, neither morphologically nor cytochemically.

A simple and quantitative method for the alkaline hydrolysis of fattyacid derivatives occurring in the lipids of mycobacteria is described. After methylation, the lipidic mixtures were chromatographed on a thin layer of silicagel. The contents in mycolates and secondary alcohols allowed the distinction of 9 groups among the 27 species studied. Such an analysis is generally insufficient to identify a species, but its discriminating power differs from that of other commonly-used methods. Complementary tests chosen according to each particular situation are necessary, including vapour phase chromatography applied to the same lipidic mixture as that used for thin-layer chromatography. Coupling of the two chromatographic methods would allow the recognition of 22 groups among the 27 species of mycobacteria studied.