Characterization and expression of secA in Mycobacterium avium.

Mycobacterium avium is both a pathogen that infects several hosts such as humans, pigs, and birds, as well as a microorganism that is encountered in environmental sources (soil and water). Protein secretion by the bacterium is likely to influence its ability to overcome adverse and competitive conditions both within or outside the host. Using a combination of cloning and information available in the databank, we characterized the secA gene from M. avium, encoding for a major preprotein translocase subunit associated with the secretion system of prokaryotics. In addition, we cloned the secA promoter sequence in a reporter construct upstream of a promoterless gfp. It was determined that the secA of M. avium shares large homology with the secA of Mycobacterium tuberculosis but not with secA of Mycobacterium leprae. secA expression was determined to be greater at logarithmic growth phase although it was also expressed at low levels during the stationary phase. secA expression was also observed when the bacteria were incubated in water as well as within human monocyte-derived macrophages and in conditions that are associated with biofilm formation. Future evaluation of the sec pathway in M. avium might provide important information about secreted proteins that are required for survival in different environments.

Potential role of roxithromycin against the Mycobacterium avium complex.

Until the recent experience with azithromycin and clarithromycin, macrolides were not considered to be important agents against mycobacteria. Clinical evidence is now growing that the newer 14 and 15 membered macrolide compounds have therapeutic activity against Mycobacterium avium, Mycobacterium chelonae and Mycobacterium leprae. Several years ago, when evaluating the activity of roxithromycin using one of the more virulent M. avium in our collection, the authors found that roxithromycin exerted a bacteriostatic effect in cultured human macrophages. However, in combination with tumour necrosis factor, which induces macrophage activation, roxithromycin caused enhanced intracellular killing. The significance of this finding is that tumour necrosis factor can be elaborated by activated macrophages during the course of infection. The roxithromycin doses that were chosen for these studies were less than achievable blood levels. More recently, the in vitro effect of roxithromycin against a panel of isolates from AIDS patients has been assessed and it was found that some (but not all) of the inhibitory concentrations, by the T-100 method of Inderlied, are within achievable serum levels. This, however, may not be the basis for anticipating in vivo activity since macrolide compounds are known to be concentrated within cells and particularly within phagolysosomes. Demonstration of effect in an in vitro test system is encouraging, but should be considered only as a preliminary step to careful assessments in experimental animals, such as the beige mouse, and studies in humans.

Recombinant cytokines for controlling mycobacterial infections.

Knowing how mycobacteria exploit host cytokines to survive and which cytokines have important roles in host defense against mycobacteria should allow the use of these molecules in the treatment of mycobacterial infections. Both interleukin 2 and interferon gamma have been used to treat patients with leprosy, and granulocyte-macrophage colony-stimulating factor is presently being administered to AIDS patients infected with Mycobacterium avium.

Use of liposome preparation to treat mycobacterial infections.

Infections caused by organisms of the genus mycobacteria, such as tuberculosis M. avium disseminated infection in AIDS patients and leprosy, are extremely common around the world. Mycobacteria are intracellular organisms that invade and multiply chiefly within phagocytic cells. Antibiotic resistance among mycobacteria is a growing concern. M. tuberculosis resistant to INH and rifampin are increasing in major urban centers of the developed and in the developing world. M. avium is characteristically resistant to most anti-tuberculosis antibiotics. Furthermore, therapy of mycobacterial infections takes a long time and most of the drugs have potential side effects and toxicity. In addition, mycobacteria is found within cells and antimicrobials need to be able to achieve adequate concentration within the compartment where mycobacteria is located. Liposome preparations, containing antibiotics, have a theoretical advantage in being able to deliver high concentrations of antimicrobials into the infected cell. Studies done thus far, in vitro and in vivo, have confirmed this premise, when comparing drug entrapped in liposomes with free drug. This paper summarizes the results obtained using liposome preparations to treat mycobacterial infections.

The role of cytokines in mycobacterial infection.

Mycobacterial infections are a major cause of morbidity and mortality worldwide. The pathogenesis of infection and the mechanisms for the development of protective immunity are poorly known, but cytokines appear to play an important role in the modulation of the immune response. Evidence exists for the role of tumor necrosis factor (TNF-alpha), granulocyte macrophage colony stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) in the host defense against mycobacteria. In this article we discuss recent findings about the role of cytokines in leprosy, tuberculosis and Mycobacterium avium infection, using in vitro and in vivo human and murine data.

Roles of soluble fibronectin and beta 1 integrin receptors in the binding of Mycobacterium leprae to nasal epithelial cells.

The mechanisms by which Mycobacterium leprae invades the human host are presently unknown. We investigated the ability of M. leprae to bind to human RPMI 2650 cells, a human nasal septal epithelial cell line, using both microscopic observation and an ELISA technique. The results demonstrated that M. leprae adheres to nasal cells after binding to soluble fibronectin. Furthermore, it was observed that M. leprae could bind to the beta 1 chain of the integrins in the absence of serum or mucus. These results demonstrated that M. leprae uses fibronectin and fibronectin receptors on the surface of epithelial cells to bind and possibly invade the nasal epithelial cells.

Postantibiotic effect of amikacin and rifapentine against Mycobacterium avium complex.

Postantibiotic effect (PAE) has received little attention in the therapy of chronic intracellular infections, such as those caused by mycobacteria. Amikacin is active therapeutically against Mycobacterium avium complex, even though serum levels exceed the MIC for only a few hours. To determine the PAE of amikacin and rifapentine for M. avium, bacteria were exposed to concentrations of 1x, 4x, and 10x the MIC of each drug for up to 120 min. Regrowth of M. avium was compared with similarly diluted untreated cultures. No PAE was observed on an inoculum of 10(4) bacteria when rifapentine was used at 5x MIC, although a slight inhibition of growth was obtained at 10x MIC for 2 h. For amikacin, PAE was observed up to 48 h at concentrations of 4x and 8x MIC and exposure times of 30-120 min. A PAE of 22 h was seen with 10(7) cfu of M. avium during incubation for 30 min with amikacin at 4x MIC. These results show that amikacin, unlike rifapentine, has a long PAE against M. avium.