Mycobacterium tuberculosis resistance in pulmonary TB patients in Cameroon: a phenotypic susceptibility assay.

OBJECTIVE: To determine the resistance of Mycobacterium tuberculosis to first- and second-line agents in adult pulmonary tuberculosis (TB) patients in Cameroon using a novel phenotypic assay. SETTING: Samples were collected from TB patients at Bamenda Hospital in Bamenda, Cameroon. DESIGN: Samples were collected consecutively from adult pulmonary TB patients over a 2-month period. TREK Sensititre(TM) MYCOTB panels were used to perform phenotypic drug susceptibility testing (DST). Susceptibility/resistance was determined by comparing minimum inhibitory concentrations to standard critical concentrations established for first- and second-line anti-tuberculosis drugs. RESULTS: Of 103 sputum samples processed, growth on Löwenstein-Jensen media was confirmed in 78 samples, 65 of which were suitable for DST. Thirty-nine strains (60%) were susceptible to all first- and second-line drugs. Five strains (8%) were categorized as multidrug-resistant TB. Two strains (3%) were classified as pre-extensively drug-resistant TB. Of those isolates susceptible to first-line drugs, 20% were resistant to at least one second-line drug. CONCLUSION: Antimicrobial resistance may be higher than assumed in TB strains in Cameroon, especially with regard to second-line drugs. There remains a need for rapid, comprehensive DST.

In vitro activity of a novel antimycobacterial compound, N-octanesulfonylacetamide, and its effects on lipid and mycolic acid synthesis.

beta-Sulfonyl carboxamides have been proposed to serve as transition-state analogues of the beta-ketoacyl synthase reaction involved in fatty acid elongation. We tested the efficacy of N-octanesulfonylacetamide (OSA) as an inhibitor of fatty acid and mycolic acid biosynthesis in mycobacteria. Using the BACTEC radiometric growth system, we observed that OSA inhibits the growth of several species of slow-growing mycobacteria, including Mycobacterium tuberculosis (H37Rv and clinical isolates), the Mycobacterium avium complex (MAC), Mycobacterium bovis BCG, Mycobacterium kansasii, and others. Nearly all species and strains tested, including isoniazid and multidrug resistant isolates of M. tuberculosis, were susceptible to OSA, with MICs ranging from 6.25 to 12.5 microg/ml. Only three clinical isolates of M. tuberculosis (CSU93, OT2724, and 401296), MAC, and Mycobacterium paratuberculosis required an OSA MIC higher than 25.0 microg/ml. Rapid-growing mycobacterial species, such as Mycobacterium smegmatis, Mycobacterium fortuitum, and others, were not susceptible at concentrations of up to 100 microg/ml. A 2-dimensional thin-layer chromatography system showed that OSA treatment resulted in a significant decrease in all species of mycolic acids present in BCG. In contrast, mycolic acids in M. smegmatis were relatively unaffected following exposure to OSA. Other lipids, including polar and nonpolar extractable classes, were unchanged following exposure to OSA in both BCG and M. smegmatis. Transmission electron microscopy of OSA-treated BCG cells revealed a disruption in cell wall synthesis and incomplete septum formation. Our results indicate that OSA inhibits the growth of several species of mycobacteria, including both isoniazid-resistant and multidrug resistant strains of M. tuberculosis. This inhibition may be the result of OSA-mediated effects on mycolic acid synthesis in slow-growing mycobacteria or inhibition via an undescribed mechanism. Our results indicate that OSA may serve as a promising lead compound for future antituberculous drug development.

Efficacies of BCG and vole bacillus (Mycobacterium microti) vaccines in preventing clinically apparent pulmonary tuberculosis in rabbits: a preliminary report.

Tuberculosis (TB) kills more people in the world today than any other infectious disease, and the number of drug-resistant Mycobacterium tuberculosis isolates is increasing. Vaccines, better than most of the currently available strains of bacille Calmette-Guérin (BCG), are urgently needed to control this disease. TB in rabbits resembles human TB more closely than TB in any other common laboratory animal and a most pertinent method of assessing vaccine efficacy is Lurie's tubercle count method in this species. Vaccinated and control rabbits were infected by aerosol with virulent human-type tubercle bacilli (H37Rv). At necropsy 5 weeks thereafter, the grossly visible primary tubercles in the entire lung were counted. A decrease in the number of such tubercles is a quantitative measure of vaccine efficacy: An effective vaccine prevents microscopic tubercles from growing to grossly visible (clinically apparent) size. The Pasteur substrain of BCG and two substrains of Mycobacterium microti (the vole bacillus) reduced the number of visible primary tubercles an average of 75%, whereas three other substrains of BCG and three other substrains of vole bacilli only reduced the number an average of 40%. These initial studies indicate that Lurie's tubercle-count method in rabbits is a precise way to choose the best available tuberculosis vaccines.

A new class of antituberculosis agents.

Long-chain lipid envelopes are characteristic of mycobacteria such as those that cause tuberculosis and leprosy. Inhibition of fatty acid synthesis or elongation is a strategy demonstrated to be clinically effective against M. tuberculosis. A new class of compounds designed to inhibit the beta-ketoacyl synthase reaction of fatty acid synthesis has been developed. Of >30 compounds described, the most active were acetamides containing alkylsulfonyl substituents. Inhibitory activities were acutely sensitive to net charge, chain length, and degree of unsaturation. The most active compound 5 (alkyl = C(10)) contained a single methylene spacer between the sulfone and carboxamide and exhibited an MIC of 0.75-1.5 microg/mL, comparable to first-line antituberculosis drugs. These compounds are species-specific, exhibiting no significant activity against bacterial species other than M. tuberculosis and closely related strains. The synthesis, biological activity, and specificity of these compounds are described.

Virulence of Mycobacterium tuberculosis CDC1551 and H37Rv in rabbits evaluated by Lurie's pulmonary tubercle count method.

The virulence of the CDC1551 strain of Mycobacterium tuberculosis was compared to that of H37Rv in a rabbit inhalation model. While rabbits that inhaled the two strains produced equal numbers of grossly visible primary tubercles, CDC1551 tubercles were smaller and contained fewer bacilli than H37Rv tubercles. These findings suggest that a miniepidemic near the Kentucky-Tennessee border caused by CDC1551 was due not to increased virulence but to increased transmissibility.

Antimycobacterial activity of cerulenin and its effects on lipid biosynthesis.

Cerulenin is a potent inhibitor of fatty acid synthase (FAS) in a variety of prokaryotic and eukaryotic cells. Using a standardized mycobacterial susceptibility test, we have observed that cerulenin inhibits the growth of several species of mycobacteria, including tuberculous species such as Mycobacterium tuberculosis (H37Rv and clinical isolates) and Mycobacterium bovis BCG (hereafter called BCG), as well as several non-tuberculous species: Mycobacterium smegmatis, the Mycobacterium avium-intracellulare complex (MAC), Mycobacterium kansasii and others. All species and strains tested, including multi-drug resistant isolates of M. tuberculosis, were susceptible to cerulenin with MICs ranging from 1.5 to 12.5 mg/L. Two-dimensional thin-layer chromatography revealed different inhibition patterns of lipid synthesis between tuberculous and non-tuberculous mycobacteria. Cerulenin treatment resulted in a relative increase in phospholipids and mycolic acids in MAC and M. smegmatis, whereas in cerulenin-treated BCG, phospholipids and mycolic acids diminished relative to controls. In addition, long-chain extractable lipids (intermediate in polarity), triglycerides and glycopeptidolipids decreased with cerulenin treatment in all three species of mycobacteria tested. Qualitative changes in several of these lipid classes indicate inhibition in the synthesis of intermediate and long-chain fatty acids. Our results suggest that cerulenin's primary effect may be inhibition of intermediate and long-chain lipid synthesis, with little effect on the synthesis of other lipid classes. In addition, the BCG-specific reduction in phospholipids and mycolic acids suggests the presence of a unique cerulenin-sensitive FAS system in tuberculous mycobacteria. Since pathogenic mycobacteria produce novel long-chain fatty acids, inhibition of fatty acid synthesis offers a potential target for the development of antimycobacterial drugs.

Mechanisms of latency in Mycobacterium tuberculosis.

Mycobacterium tuberculosis can persist within the human host for years without causing disease, in a syndrome known as latent tuberculosis (TB). As one-third of the world population has latent TB, placing them at risk for active TB, the mechanisms by which M. tuberculosis establishes a latent metabolic state, eludes immune surveillance and responds to triggers that stimulate reactivation are a high priority for the future control of TB.

Brachial-plexus palsy in the newborn.

Twenty-one patients who had twenty-three brachial-plexus palsies resulting from trauma at birth were studied over a three and one-half year period, between July 1983 and December 1986. The incidence of this group of injuries was 2.5 per 1,000 live births. There were fourteen palsies of the fifth and sixth cervical nerves; eight of the fifth, sixth, and seventh cervical nerves; and one of the entire brachial plexus. Two patients were lost to follow-up shortly after birth. Of the remaining nineteen patients (twenty-one palsies), fifteen (seventeen palsies) had full recovery at an average of three months (range, two weeks to twelve months), and four (four palsies) had residual paralysis of the upper extremity at more than twenty-six months. We concluded that the newborn who has a brachial-plexus palsy has a favorable prognosis for complete recovery.