Stazyme, a mycobacteriolytic preparation from a Staphylococcus strain, is able to break the permeability barrier in multiple drug resistant Mycobacterium avium.

As a strategy to augment the potential of existing drugs against Mycobacterium avium we investigated a mycobacteriolytic preparation (stazyme) from the Staphylococcus strain Clavelis, which results in significant mycobacterial growth inhibition. A total of 10 specific protein bands were characterized in the stazyme preparation: three bands within a major 40-60 kDa fraction, five bands within the range of 30-90 kDa, and two bands of about 12 and 14 kDa respectively. Tested at concentrations of 50 and 200 microg ml(-1) of total protein, stazyme was highly bactericidal against M. smegmatis, and bacteriostatic against M. tuberculosis and M. avium. Stazyme was able to break the permeability barrier of M. avium isolates, significantly enhancing the activity of other antituberculous drugs (ethambutol, rifampicin, and amikacin), used at sub-MIC level. Stazyme essentially possessed a lytic activity as evidenced by its ability to lyse purified M. smegmatis cell walls. This lytic activity was also confirmed on intact M. smegmatis and M. avium bacilli by transmission electron microscopy. Precise identification of this mycobacteriolytic determinant(s) in stazyme may be helpful to define novel drug targets in mycobacteria.

Emergence during unsuccessful chemotherapy of multiple drug resistance in a strain of Mycobacterium tuberculosis.

Serial isolates of Mycobacterium tuberculosis were cultured from a patient who failed to respond to standard antituberculous chemotherapy. Isolates were cultured in March 1989, July 1989, December 1989 and May 1990. Each successive isolate was found to be resistant to a wider range of antituberculous drugs than its predecessors. The initial isolate was resistant to isoniazid and rifampin, the second isolate was also resistant to ethambutol, the third was also resistant to pyrazinamide, ansamycin (= rifabutin) and ofloxacin and the last isolate was also resistant to ciprofloxacin and sparfloxacin. All four isolates' bacteriophage typing profiles and DNA restriction fragment patterns determined by Southern blot hybridization using the IS6110/IS986 probes and the new probe pTBN12 were concordant. It was concluded that this patient was persistently infected with a single strain of Mycobacterium tuberculosis which developed resistance to a number of families of drugs but did not show any significant change in typing patterns. The problem of acquired multiple drug resistance, particularly to fluoroquinolones and rifamycins, represents a new challenge in tuberculosis therapy.

Cloning and expression of Mycobacterium aurum carotenogenesis genes in Mycobacterium smegmatis.

This report describes the first successful transfer and complete expression of clustered mycobacterial genes controlling a biosynthetic pathway (carotenogenesis) in a homologous system. A genomic library of pigmented Mycobacterium aurum A+ (yellow-orange) DNA was constructed in shuttle vector pHLD-69. The colourless mutant A11 and the brick-red mutant NgR9 derived from M. aurum A+ were electroporated with the plasmid library. Among the transformants, colonies different in colour from the recipient mutants were detected, and were cloned. One of the clones from the transformed A11 mutant had a yellow-orange phenotype, and was designated A11T; one of the clones from the NgR9 (brick-red) mutant had a yellow-orange phenotype and was designated NgR9T. The carotenoid pigments from the A11T and NgR9T clones were analyzed and in both the end product of carotenogenesis in M. aurum (leprotene) was detected. A11T and NgR9T harboured the same recombinant plasmid (Cl) containing a 11-kb M. aurum fragment. pCl was used to transform the colourless Mycobacterium smegmatis MC2-155 strain. All the transformants were pigmented. A colony (MC2-T) was arbitrarily chosen and leprotene was detected. It was therefore concluded that M. aurum genes involved in carotenogenesis had been cloned, and were expressed not only in M. aurum mutants, but also in M. smegmatis.

Conjugative transfer of a shuttle plasmid from Escherichia coli to Mycobacterium smegmatis [corrected].

The chimeric plasmid pMY10 containing the origin of replication of pAL5000, the origin of replication of pBR322, the origin of transfer of pRK2 and a kanamycin resistance gene was constructed and successfully transferred by conjugation from Escherichia coli harbouring the helper plasmid pRK4.24 into Mycobacterium smegmatis. This is the first report of conjugtive transfer of plasmid between E. coli and an acid fast organism.

Restriction map of mycobacteriophage D29 and its deletion mutant F5.

A physical map of mycobacteriophage D29 was constructed, including positions for 25 restriction sites for 9 endunocleasic enzymes. D29 DNA contains about 48 150 bp. Analysis of a deletion mutant (F5) has allowed to determine the location of two non essential regions in the genome, allowing further insertion of foreign genes and construction of cosmids.

Methionine as methyl-group donor in the synthesis of Mycobacterium avium envelope lipids, and its inhibition by DL-ethionine, D-norleucine and DL-norleucine.

The radioactivity from 3H-methyl methionine was rapidly incorporated into the surface lipids of Mycobacterium avium. The transmethylation reaction was efficiently inhibited by DL-ethionine, D-norleucine and DL-norleucine. The structure of the outerlayer of the M. avium envelope was profoundly altered in the bacteria treated with DL-norleucine.

[Distribution of bacteriophage types of Mycobacterium tuberculosis in France]. / Répartition des lysotypes de Mycobacterium tuberculosis en France.

The bacteriophage typing of M. tuberculosis enables separation of the strains of the species according to their sensitivity to certain bacteriophages. A relationship has been observed between the geographical origin of patients and the distribution of the phage types of their strains. This study related to a sample of 450 strains isolated between 1978 and 1987 in patients of French origin coming from different regions in France. An analysis of the data provided by the study indicates that the distribution of the 5 phage types, A, AX, I, B, and C is relatively homogenous in the different regions with a predominance of the phage type A and B. The mean percentage of the lysotypes A, AX, I, B and C in the country as a whole are 43%, 15%, 7%, 30% and 5% respectively. Only in region 3 (Haute and Basse Normandie), 8 (Provence-Côte-d'Azur) and 9 (Rhône-Alpes) is there any perceptible deviation from this distribution. This study also underlines the contribution that can be provided by this type of information in the epidemiology of tuberculosis.

Studies on clofazimine-resistance in mycobacteria: is the inability to isolate drug-resistance mutants related to its mode of action?

This study showed that clofazimine was not radiomimetic, it was not a mutagenic compound, it was not an inducer of prophage-lambda, and it did not eliminate plasmids from appropriate host bacteria. The drug caused an effective inhibition of the growth of Mycobacterium aurum, and also inhibited the growth cycle of the mycobacteriophage D29. Cross-resistance between clofazimine, streptomycin and rifampicin could not be demonstrated. Drug-resistance mutants towards clofazimine could not be isolated, and it was found that the existing clofazimine-resistant strains of Mycobacterium tuberculosis were rather susceptible organisms requiring clofazimine in their growth medium to maintain their drug-resistance. Ultrastructural studies showed that clofazimine did not act by cell wall lysis, nor did it act on bacterial ribosomes. Higher concentrations of the drug resulted in bacterial plasmolysis. These findings are discussed in the light of its known properties and proposed mode of action.

Uptake of selected antibacterial agents in Mycobacterium avium.

The antibacterial action of 64 drugs against Mycobacterium avium ATCC 15769 was screened in Middlebrook 7H9 liquid medium. The most active drugs were ansamycin, rifampicin, clofazimine, and pristinamycin. The antibacterial action of the selected drugs was confirmed by testing clinical isolates on Middlebrook 7H10 agar medium. The antibacterial actions were not related with the hydrophobicities and molecular weights of the drugs. However, all the active drugs were highly hydrophobic molecules of low polarity. These drugs dissolved into the lipids forming the outer layers of the bacterial envelope and they appeared to interact with the surface amphiphils.

Structure of the cell envelope of Mycobacterium avium.

In this report the cell wall of Mycobacterium avium is shown as a triple-layered structure where the outermost layer was stained by the ruthenium red staining for polysaccharides. The outermost layer hindered the diffusion of chemotherapeutic agents across the wall thus causing multiple drug-resistance by exclusion. The concerted electron microscopy and chemical analysis of chloroform-methanol and Triton X-100 extracts indicated that the outer layer was made of diverse amphiphil glycolipids (mycosides C, glycolipids, peptidolipids, phospholipids) that formed a matrix in which proteins were embedded. The examination of a spontaneous rough mutant indicated that mutations blocking the synthesis of one or several of the amphiphils must leave unsubstituted mycolic acid residues, thus causing surface hydrophobicity and roughness. Judging from our data, a model describing the overall cell envelope of M. avium was proposed. From the comparative analysis of M. avium, its spontaneous rough mutant, and its spheroplasts, some of the functions of the outermost layer were disclosed.

Action of colistin (polymyxin E) on the lytic cycle of the mycobacteriophage D29 in Mycobacterium tuberculosis.

The antibiotic colistin (polymyxin E) inhibited the lytic cycle of the mycobacteriophage D29 in the tubercle bacilli, but not the D29 adsorption. The protein and nucleic acid synthesis in D29-infected bacteria were not affected significantly. The inhibitory activity was reversed by washing off the antibiotic, and by addition of Ca++, but not in media made iso-osmotic by addition of NaCl or sucrose. Transmission electron microscopy revealed an asymmetric to symmetric transition in the staining profile of the cytoplasmic membrane. Though no mature phage particles were ever observed in colistin-treated, D29-infected tubercle bacilli, loosely arranged aggregates resembling phage proheads were occasionally found. Judging from the above data, it was concluded that colistin inhibited D29 lytic cycle by causing molecular displacements in the inner leaflet of the cytoplasmic membrane, and consequently, the binding sites for D29 structural proteins were not available.

Abortive infection of Mycobacterium leprae by the mycobacteriophage D29.

The interactions of mycobacteriophage D29 and Mycobacterium leprae were examined. It was demonstrated that after adsorption D29 injected its DNA in M. leprae. While the synthesis of host proteins and lipids were inhibited in M. tuberculosis and in M. smegmatis during infection by D29, the results were inconclusive in the case of M. leprae because these bacteria did not incorporate the appropriate substrates.

[Distribution of lysotypes of Mycobacterium tuberculosis in relation to the country of origin of the patient]. / Répartition des lysotypes de Mycobacterium tuberculosis en relation avec le pays d'origine du malade.

The diversity among the phage types of 422 strains of Mycobacterium tuberculosis isolated from patients of different geographical origins suggested that this method might prove useful in studying the propagation of tuberculosis in different types of populations. We first investigated M. tuberculosis strains isolated from France, Portugal, Romania, Algeria, Egypt, Uganda, Mali and India. We then studied in detail two groups of immigrants residing in France, from Portugal and North Africa, respectively. This investigation showed that most patients were suffering from a M. tuberculosis strain with a phage type specific of their country of origin. Factor analysis helped to display phage typing relationships with respect to the geographical origin of patients.