Rifamycin Antibiotics Research Articles

Discovery of a New Source of Rifamycin Antibiotics in Marine Sponge Actinobacteria by Phylogenetic Prediction

Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora strains of actinobacteria indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the rifamycin B synthase of Amycolatopsis mediterranei. This result was not expected from taxonomic species tree phylogenetics using 16S rRNA sequences. From the PKS sequence data generated from our sponge-derived Salinispora strains, we predicted that such strains might synthesize rifamycin-like compounds. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of rifamycin synthesis. The analysis reported here demonstrates that this Salinispora isolate does produce compounds of the rifamycin class, including rifamycin B and rifamycin SV. A rifamycin-specific KS primer set was designed, and that primer set increased the number of rifamycin-positive strains detected by PCR screening relative to the number detectable using a conserved KS-specific set. Thus, the Salinispora group of actinobacteria represents a potential new source of rifamycins outside the genus Amycolatopsis and the first recorded source of rifamycins from marine bacteria.

Normal and reversed-phase TLC separations of some macrocyclic antibiotics with non-aqueous mobile phases

The chromatographic behavior of five macrocyclic antibiotics (erythromycin, troleandomycin, tylosin, rifamycin B, and rifampicin) has been compared on normal and reversed-phase TLC plates. Mobile phases were wide-ranging mixtures containing from 0 to 100%, ( v/v ), esters or ketones in dimethyl sulfoxide (DMSO) or hexamethyldisiloxane (HMDSO). Chromatographic retention data and a possible retention mechanism are discussed.

Inhibition of mutation and combating the evolution of antibiotic resistance.

The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.

Targeted mutagenesis of the Mycobacterium smegmatis mca gene, encoding a mycothiol-dependent detoxification protein.

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.

Analysis of selected macrocyclic antibiotics by HPTLC with non-aqueous binary mobile phases

The retention behavior of selected macrocyclic antibiotics (erythromycin, troleandomycin, tylosin, vancomycin, rifamycin B, and rifampicin) has been examined on LiChrospher Si 60 F 254s HPTLC plates as stationary phase. A wide range of mixtures of alcohols and ketones with hexamethyldisiloxane (HMDSO) in proportions from 0 to 100% ( v/v ) and with dimethyl sulfoxide (DMSO) in proportions from 0 to 50% ( v/v ) were used as mobile phases. Chromatographic retention data and a possible retention mechanism are discussed.

Mycobacterial polyketide-associated proteins are acyltransferases: proof of principle with Mycobacterium tuberculosis PapA5.

Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as well as PapA5 homologs found in Mt and other species, are a subfamily of acyltransferases. Studies with recombinant protein confirmed that PapA5 is an acyltransferase [corrected]. Deletion analysis in Mt demonstrated that papA5 is required for PDIM synthesis. We propose that PapA5 catalyzes diesterification of phthiocerol and phthiodiolone with mycocerosate. These studies present the functional characterization of a Pap and permit inferences regarding roles of other Paps in the synthesis of complex lipids, including the antibiotic rifamycin.

A Switch for the transfer of substrate between nonribosomal peptide and polyketide modules of the rifamycin synthetase assembly line.

A nonribosomal peptide synthetase (NRPS) loading module and a polyketide synthase (PKS) elongation module catalyze the preliminary steps in the biosynthesis of the rifamycin antibiotics. A benzoate molecule is covalently attached to the phosphopantetheine arm of the thiolation domain of the loading module when its reaction partner methylmalonyl-CoA is absent. Occupancy of the thiolation domain of the elongation module by a methylmalonyl moiety appears to trigger intermodular transfer of benzoate to the ketosynthase domain of the elongation module. This transthiolation event is fast relative to the initial loading of benzoate onto the loading module. It will be of interest to determine if these results are generally true for intermodular acyl transfer in other NRPS-PKS and PKS assembly lines.

Rifamycin antibiotics—new compounds and synthetic methods. Part 1: Study of the reaction of 3-formylrifamycin SV with primary alkylamines or ammonia

Within the study covering the search for new methods of synthesis of rifamycin antibiotics, the reactions of 3-formylrifamycin SV ( 2 ) with primary amines or ammonia were studied. In the reaction of 2 with methylamine, unstable 3-methyliminomethylrifamycin SV ( 8 ) was formed, which was further oxidised to stable 3-methyliminomethylrifamycin S ( 9 ). In the reaction of 2 with ammonia, N,15-didehydro-15-deoxo-pyrimido-(4,5- b)rifamycin SV ( 10 ), a new compound with a chromophore system enlarged by a pyrimidine ring, was obtained. The product of its reduction with sodium borohydride—( 11 )—was also isolated. The structures of the compounds and an explanation of the synthesis mechanism have been proposed on the basis of mass spectrometry results as well as (1D) and (2D) 1H- and 13C NMR analysis. In vitro antituberculous activity of the new compounds have been investigated.

Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver

The antibiotics rifamycin SV and rifampicin substantially reduce sulfobromophthalein (BSP) elimination in humans. In rats, rifamycin SV and rifampicin were shown to interfere with hepatic organic anion uptake by inhibition of the organic anion transporting polypeptides Oatp1 and Oatp2. Therefore, we investigated the effects of rifamycin SV and rifampicin on the OATPs of human liver and determined whether rifampicin is a substrate of 1 or several of these carriers. In complementary RNA (cRNA)-injected Xenopus laevis oocytes, rifamycin SV (10 μmol/L) cis-inhibited human organic anion transporting polypeptide C ( SLC21A6) (OATP-C), human organic anion transporting polypeptide 8 ( SLC21A8) (OATP8), human organic anion transporting polypeptide B ( SLC21A9) (OATP-B), and human organic anion transporting polypeptide A ( SLC21A3) (OATP-A) mediated BSP uptake by 69%, 79%, 89%, and 57%, respectively, as compared with uptake into control oocytes. In the presence of 100 μmol/L rifamycin SV, BSP uptake was almost completely abolished. Approximate K i values were 2 μmol/L for OATP-C, 3 μmol/L for OATP8, 3 μmol/L for OATP-B and 11 μmol/L for OATP-A. Rifampicin (10 μmol/L) inhibited OATP8-mediated BSP uptake by 50%, whereas inhibition of OATP-C–, OATP-B–, and OATP-A–mediated BSP transport was below 15%. 100 μmol/L rifampicin inhibited OATP-C– and OATP8-, OATP-B– and OATP-A–mediated BSP uptake by 66%, 96%, 25%, and 49%, respectively. The corresponding K i values were 17 μmol/L for OATP-C, 5 μmol/L for OATP8, and 51 μmol/L for OATP-A. Direct transport of rifampicin could be shown for OATP-C (apparent K m value 13 μmol/L) and OATP8 (2.3 μmol/L). In conclusion, these results show that rifamycin SV and rifampicin interact with OATP-mediated substrate transport to different extents. Inhibition of human liver OATPs can explain the previously observed effects of rifamycin SV and rifampicin on hepatic organic anion elimination. (H EPATOLOGY 2002;36:164-172.)

Active Transport of an Antibiotic Rifamycin Derivative by the Outer-Membrane Protein FhuA

Background: FhuA, an integral membrane protein of Escherichia coli, actively transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane. The transport is coupled to the proton motive force, which energizes FhuA through the inner-membrane protein TonB. FhuA also transports the semisynthetic rifamycin derivative CGP 4832, although the chemical structure of this antibiotic differs markedly from that of ferric hydroxamates. Results: X-ray crystallography revealed that rifamycin CGP 4832 occupies the same ligand binding site as ferrichrome and albomycin, thus demonstrating a surprising lack of selectivity. However, the binding of rifamycin CGP 4832 is deviant from the complexes of FhuA with hydroxamate-type ligands in that it does not result in the unwinding of the switch helix but only in its destabilization, as reflected by increased B factors. Unwinding of the switch helix is proposed to be required for efficient binding of TonB to FhuA and for coupling the proton motive force of the cytoplasmic membrane with energy-dependent ligand transport. The transport data from cells expressing mutant FhuA proteins indicated conserved structural and mechanistic requirements for the transport of both types of compounds. Conclusions: We conclude that the binding of rifamycin CGP 4832 destabilizes the switch helix and promotes the formation of a transport-competent FhuA-TonB complex, albeit with lower efficiency than ferrichrome. Active transport of this rifamycin derivative explains the 200-fold increase in potency as compared to rifamycin, which is not a FhuA-specific ligand and permeates across the cell envelope by passive diffusion only.

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.

The Importance of Homologous Recombination in the Generation of Large Deletions in Hybrid Plasmids in Amycolatopsis mediterranei

The cloning vector pRL60 was developed previously as a tool for genetic manipulations in Amycolatopsis mediterranei, which produces the commercially and medicinally important antibiotic rifamycin. Here, a method based on intraplasmid recombinations is described for the construction of smaller plasmids in A. mediterranei, which also helped in delimiting the origin of replication (pA-rep) of the parent plasmid. The strategy involved the cloning of a selectable marker, erythromycin resistance gene (ermE), onto plasmids pULAM2 and pULVK2A (derivatives of pRL1), followed by selection of the hybrid or concatemeric plasmids pRL50 and pRL80 (with large homologous repeats) in Escherichia coli GM2163. These hybrid plasmids were then transferred to A. mediterranei DSM 40773 by electroporation, with selection in the presence of different antibiotics. During the process of transformation and selection in A. mediterranei, pRL50 and pRL80 underwent intraplasmid recombinations, yielding derivatives that retained a common region essential for maintenance and replication, as well as the selected resistance genes. This approach produced several smaller plasmids designated pRL51, pRL52, pRL53, pRL60, pRL81, and pRL82. These plasmids, isolated from A. mediterranei DSM 40773, could be transferred to different Amycolatopsis strains at transformation efficiencies ranging from 0.7 × 102 to 4 × 104 transformants/μg DNA. The electroporation parameters under which maximum transformation efficiencies were obtained varied from strain to strain. Since the isolation of plasmid DNA from Amycolatopsis strains were extremely difficult, a convenient and rapid method of direct transfer of plasmid DNA, i.e., electroduction, was also developed in which the above-described shuttle plasmids were transferred directly from A. mediterranei to E. coli. In addition, the sequence of the minimal (pA-rep, ∼1.0 kb) of plasmid pRL51 was determined. The nucleotide base sequence of the pA-rep region did not have any clear similarity to the DNA or amino acid sequences in various databases, suggesting that it is unique.

Self-Association of Rifamycin B: Possible Effects on Molecular Recognition

The macrocyclic antibiotic rifamycin B was found to be highly surface-active and to aggregate in aqueous solution. The aggregational behavior was studied using small-angle neutron scattering (SANS). Aqueous solutions of rifamycin B showed pronounced scattering at both small-length scales (Q ≥ 0.1 Å-1) and at large-length scales (Q ≤ 0.1 Å-1). The larger association colloids appear to be rather open low-density aggregates. The addition of 10% 2-propanol greatly reduces the number and size of the aggregates. Somewhat higher amounts of alcohol appear to completely suppress or eliminate aggregation. The suppression of aggregation coincides with the appearance and enhancement of enantioselective association between rifamycin B and a variety of chiral amino alcohols. It appears that the self-aggregation of rifamycin B may be a factor that controls its ability to differentiate between enantiomers in aqueous and hydro-organic solutions.

Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699

The ansamycin class of antibiotics are produced by various Actinomycetes. Their carbon framework arises from the polyketide pathway via a polyketide synthase (PKS) that uses an unusual starter unit. Rifamycin (rif), produced by Amycolatopsis mediterranei, is the archetype ansamycin and it is medically important. Although its basic precursors (3-amino-5-hydroxy benzoic acid AHBA, and acetic and propionic acids) had been established, and several biosynthetic intermediates had been identified, very little was known about the origin of AHBA nor had the PKS and the various genes and enzymes that modify the initial intermediate been characterized. A set of 34 genes clustered around the rifK gene encoding AHBA synthase were defined by sequencing all but 5 kilobases (kb) of a 95 kb contiguous region of DNA from A. mediterranei. The involvement of some of the genes in the biosynthesis of rifamycin B was examined. At least five genes were shown to be essential for the synthesis of AHBA, five genes were determined to encode the modular type I PKS that uses AHBA as the starter unit, and 20 or more genes appear to govern modification of the polyketide-derived framework, and rifamycin resistance and export. Putative regulatory genes were also identified. Disruption of the PKS genes at the end of rifA abolished rifamycin B production and resulted in the formation of P8/1-OG, a known shunt product of rifamycin biosynthesis, whereas disruption of the orf6 and orf9 genes, which may encode deoxysugar biosynthesis enzymes, had no apparent effect. Rifamycin production in A. mediterranei is governed by a single gene cluster consisting of structural, resistance and export, and regulatory genes. The genes characterized here could be modified to produce novel forms of the rifamycins that may be effective against rifamycin-resistant microorganisms.

Rifapentine.

Rifapentine is a rifamycin antibiotic with antimycobacterial activity. Rifapentine is generally more active against Mycobacterium tuberculosis than rifampicin (rifampin), although strains resistant to rifampicin are usually cross-resistant to rifapentine. Sputum culture conversion rates were slightly higher after 6 months of rifapentine- versus rifampicin-based therapy in patients with pulmonary tuberculosis in a Western study; however, relapse rates were higher in rifapentine recipients during follow-up. The excess relapses in the rifapentine group appeared to be related to poor compliance with nonrifamycin antituberculosis drugs during the intensive phase (first 2 months) of therapy. Rifapentine- and rifampicin-containing regimens produced similar sputum culture conversion rates with low rates of relapse in 2 randomised clinical trials in patients with smear-positive tuberculosis in China. In one trial, there was no difference in sputum culture conversion rates in patients treated with rifapentine once weekly or rifampicin twice weekly in combination with isoniazid and ethambutol during the continuation phase of treatment. Hyperuricaemia, which was reported only during the intensive phase, elevated ALT and AST levels and neutropenia were the most common treatment-related adverse events reported in patients receiving rifapentine- or rifampicin-containing regimens for tuberculosis in 1 Western study.

Adaptive acquisition of novobiocin resistance in Pasteurella multocida strains of avian origin.

Naturally occurring strains of Pasteurella multocida are atypically susceptible to hydrophobic antibiotics such as novobiocin, despite their Gram-negative cell envelope ultrastructure. Four strains adaptively resistant to 1000 micrograms/ml of novobiocin were obtained by sequentially subculturing cell surface hydrophobic variants of avian origin in the presence of increasing antibiotic concentrations. Adaptive novobiocin resistance was accompanied in all cases by the concomitant acquisition of resistance to coumermycin, a hydrophobic antibiotic possessing the same mechanism of action, but not to the functionally disparate hydrophobic antibiotic rifamycin. The acquisition of resistance was not accompanied by alterations in the lipid composition of the cell envelope. Subsequent growth of adaptively resistant strains in the absence of novobiocin did not result in the restoration of susceptibility to either novobiocin or coumermycin. Acquisition of adaptive resistance in encapsulated parental strains resulted in an inability to synthesize capsular material and enhanced cell surface hydrophobicity; however, parental encapsulation and decreased cell surface hydrophobicity were restored upon removal of novobiocin. These data suggest that acquisition of adaptive resistance to novobiocin conferred in this manner is the result of a stable genetic event affecting the mechanistic target of both novobiocin and coumermycin rather than a physiological adaptation involving outer membrane impermeability.

Rifaximin suspension for the eradication of Helicobacter pylori

The in vitro sensitivity of Helicobacter pylori to rifaximin, a new rifamycin antibiotic, was evaluated in 40 clinical isolates by the agar dilution method. Rifaximin showed good activity, with a 50% minimum inhibitory concentration of 4 mg/L −1. Consequently, we assessed rifaximin in H pylori—positive patients. Overall, 71 patients with upper gastrointestinal symptoms (35 men and 36 women; aged 19 to 73 years, mean, 45.6 years) were found to have H pylori—associated gastritis. The first 30 consecutive patients received monotherapy with rifaximin 600 mg three times a day (TID) for 14 days. The 41 patients enrolled thereafter were allocated in an open, randomized fashion to four different treatment groups for 14 days: (1) rifaximin 600 mg TID and colloidal bismuth subcitrate 240 mg twice a day (BID) (n = 10); (2) rifaximin 600 mg TID and omeprazole 20 mg BID (n = 10); (3) rifaximin 600 mg TID and amoxicillin 1 g BID (n = 10); or (4) rifaximin 1800 mg TID and metronidazole 500 mg TID (n = 11). Upper gastrointestinal symptoms (pyrosis, bloating, epigastric pain, and nausea) were recorded and assessed before and 4 weeks after treatment. Patients were assessed by endoscopy, histology, CP test, culture, and serology (immunoglobulin G [IgG] to H pylori) at entry. Sixty-seven patients were available for follow-up 4 weeks after the completion of treatment. A statistically significant improvement in symptoms was seen in patients treated with rifaximin and rifaximin plus colloidal bismuth subcitrate. No statistically significant differences in degree of improvement in endoscopic and histologic findings were seen among the five treatment groups. A statistically significant decrease in the mean IgG value after treatment was found for rifaximin, rifaximin plus colloidal bismuth subcitrate, and rifaximin plus omeprazole. The overall eradication rate was 43%. These results suggest that rifaximin may be an effective antibiotic against H pylori infection and is worthy of further study.

Enantiomeric resolution using the macrocyclic antibiotics rifamycin B and rifamycin SV as chiral selectors for capillary electrophoresis

Rifamycin B and rifamycin SV belong to the class of macrocyclic antibiotics known as ansamycins. These macrocyclic antibiotics were used as chiral selectors in capillary electrophoresis to enantioselectively resolve a number of chiral compounds. They contain groups capable of providing the types of multiple interactions necessary to achieve chiral recognition between enantiomers. In fact, they appear to be complimentary in the types of compounds they can enantiomerically resolve. Rifamycin B is shown to be enantioselective towards positively charged compounds, while rifamycin SV was enantioselective towards negatively charged solutes. The choice of wavelength for detection significantly affects sensitivity. Monitoring one of the wavelengths which coincide with the absorption minima of the chiral selector enhances sensitivity. Resolution is enhanced by keeping the amount of analyte injected on column as low as possible and it is demonstrated that it is possible to detect as little as 0.1% of one enantiomer in the presence of the other enantiomer using indirect detection.

The ansamycins: hypolipidemic agents stimulating cholesterol removal by nonclassical mechanisms.

The ansamycins CGP 43371 and CGS 24565 are derivatives of the antibiotic rifamycin that reduce plasma cholesterol levels in both primate and nonprimate species. In vivo, a striking accumulation of macrophage cholesteryl ester was seen in ansamycin-treated rats and hamsters, but carbon clearance studies and reticuloendothelial system blockade by gadolinium chloride indicated that phagocytosis was not involved. Simple addition of an ansamycin to macrophages or monocytes in vitro failed to stimulate radiolabeled lipoprotein cholesteryl ester association or mass accumulation. In contrast to mononuclear cells, however, the ansamycins did enhance radiolabeled lipoprotein cholesteryl ester association by liver cells in vitro. Primary hepatocyte cultures prepared from rats treated with radiolabeled CGP 43371 secreted CGP 43371 over an 18-h period in a fraction floating at d < 1.02 g/ml after density gradient ultracentrifugation that was relatively enriched in apoA-I. The medium containing this secreted [14C]GP 43371-labeled lipoprotein was capable of enhancing the cholesteryl ester content of macrophages in vitro, suggesting that ansamycin-induced liver modification of lipoproteins might be involved. These drugs may serve as valuable tools for studying mechanisms of lipoprotein uptake.

Polymerase chain reaction based diagnostic assay in diagnosis of tuberculosis

In the fourth part of an ongoing study dealing with the search for new rifamycin antibiotics, the reaction of 3-formylrifamycin SV (1) with secondary amines (a.o. pyrrolidine) and ketones of general structure R1–CH2–CO–R2 (R1=H or alkyl and R2=alkyl or aryl) has been investigated. A new synthetic method for the preparation of stable zwitterionic rifamycin derivatives with α,β-unsaturated pyrrolidinium substituents at C3 of the naphthol chromophore has been developed. The structures of the isolated microcrystalline rifamycins and a general mechanism have been proposed on the basis of mass spectrometry results, FTIR, 1D and 2D NMR as well as TLC methods. Rifamycins displaying an aromatic substituent within the pyrrolidinium fragment at C3 displayed a good stability against hydrolysis and proved, in preliminary tests, to be active against TB and MOTT, the activity being for some particular strains (Myc. tbc. Bovis and Myc. scrofulaceum) higher than that observed with the reference drugs (RMP, RBT).