Previously established radiometric techniques were used to assess the effectiveness of combined antimicrobial drug-inhibitory drug (drug-inhibitor) treatment on two clinical isolates of the Mycobacterium avium complex representing three colony variants: smooth opaque (dome) (SmO), smooth transparent (SmT), and rough (Rg). All variants were identified as members of the M. avium complex; however, only the SmT colony type of strain 373 possessed characteristic serovar-specific glycopeptidolipid (GPL) antigens. MICs, determined radiometrically, of drugs with the potential to inhibit the biosynthesis of GPL antigens or other cell envelope constituents were similar for all strains. These drugs included cerulenin, N-carbamyl-DL-phenylala- 581-2139. Fax: (205) 581-2877. properties (3,9,27) and may play a role in pathogenicity, in addition to their possible contribution to drug resistance.In an effort to design effective drug therapy for the treatment ofM. avium, we used a combined drug-inhibitor treatment that would potentially inhibit GPL biosynthesis, thereby affecting cell envelope architecture and thus allowing more effective use of antimicrobial agents that have demonstrated efficacy in M. avium therapy. For this strategy, we used drugs that have the potential to affect GPL biosynthesis at three sites: glycosylation, peptide biosynthesis, and fatty acid biosynthesis. We have reported that this approach is capable of producing a synergistic effect when drugs which potentially inhibit GPL biosynthesis are used in combination with amikacin, sparfloxacin, and clarithromycin (5). Those studies were an extension of an earlier report of combined treatment with m-fluoro-phenylalanine, an inhibitor GPL biosynthesis (14), and ethambutol, an inhibitor of arabinogalactan biosynthesis (36). That combination was effective in producing synergistic growth inhibition of M. avium (32).In our most recent publication regarding this combined drug-inhibitor treatment we used laboratory-maintained isolates: a smooth opaque (dome) (SmO) and a rough (Rg) variant of M. avium serovar 4 (5). In this report we extend our observations to a clinical isolate which presented with the SmO colony morphology upon initial isolation on LowensteinJensen medium but, upon successive subculturing on 7H9 agar, segregated into distinct colony morphotypes: smooth transparent (SmT), SmO, and Rg. All three morphotypes were identified as members of the M. avium complex. We also examined the effects of combined drug-inhibitor treatment on another Rg clinical isolate to confirm the difference in susceptibility that we observed for Rg and Sm colony variants. Internal radiolabeling techniques were used to confirm the presence or

Smooth-and rough-colony variants of Mycobacterium avium serovar 4 were treated with three classes of drugs. The drugs were chosen for their potential inhibitory effects on the biosynthesis of the cell envelopeassociated serovar-specific glycopeptidolipid antigens. Growth was monitored radiometrically with a BACTEC 460-TB instrument, and MICs were determined for each drug. Both variants were then treated with inhibitory drugs in combination with antimicrobial agents that have demonstrated effectiveness against M. avium. No growth inhibition was observed with 6-fluoro-6-deoxy-D-glucose or avidin. Inhibitors of glycosylation, i.e., 2-deoxy-D-glucose, bacitracin, and ethambutol, were inhibitory to smooth-and rough-colony variants, whereas drugs that inhibit peptide synthesis, i.e., N-carbamyl-L-isoleucine and m-fluoro-phenylalanine, were more inhibitory for the rough-colony variant. Cerulenin, which affects fatty acid synthesis, was inhibitory for both variants, but it appeared to be more effective at inhibiting the growth of the smooth-colony variant at equivalent concentrations. Generally, when inhibitors of glycosylation were used with sparfloxacin and amikacin, a synergistic effect was observed for only the smooth variant. When drugs that affect peptide synthesis were used in combination with amikacin, a synergistic effect was observed for the rough variant, and when cerulenin was used in combination with sparfloxacin or amikacin, a synergistic effect was observed for both variants. Lipid analysis revealed that although the rough variant lacks the serovar-specific glycopeptidolipid antigens, it does possess a group of phenylalanine-isoleucine-containing lipopeptides that may explain its different susceptibility patterns to m-fluoro-phenylalanine and N-carbamyl-L-isoleucine. The significance of these results is discussed with reference to various components in the cell envelope and their importance in cell wall permeability.The new emphasis of the National Institute of Allergy and Infectious Diseases is "to spark efforts" to better understand the opportunistic infections associated with AIDS so that new and effective treatments can be developed (45). A major obstacle preventing the development of better therapies for those opportunistic pathogens is a "basic lack of knowledge about the pathogens that cause such diseases" (45). The Mycobacterium avium complex represents one of the most important groups of opportunistic pathogens infecting patients with AIDS (23,45 Rastogi et al. (33) proposed that a polysaccharide outer layer may be responsible for the impermeability of smoothtransparent variants to various substrates and drugs and also increased pathogenicity. The presence of the fibrillar material described by Draper (13) and Kim et al. (21) and later referred to as the superficial L, layer (2) may also be important as a barrier because of the polar glycopeptidolipids (GPLs) that make up its superficial location (4, 6, 44). Crowle et al. (9) suggested that both the polysaccharide layer (32, 33) and the GPL layer...

The Mono Mac 6 (MM6) human monocytic cell line was evaluated with the established J774 murine macrophage cell line to ascertain its effectiveness in determining the intracellular activities of antimycobacterial drugs. Cells were infected with Mycobacterium tuberculosis H37Ra and treated with drug concentrations corresponding to the MICs, as well as to threefold higher than and threefold less than the MICs. Changes in CFU were compared after 7 days to determine significant differences between treated and nontreated groups. The results suggest that MM6 will make a useful model for testing the intracellular activities of antituberculosis drugs.

Mycobacterial fractions, some ofwhich are associated with the cell envelope ofMycobacterium avium serovar 4, were assessed for their ability to affect various immunological functions of human peripheral blood mononuclear cells (PBM). Treatment of PBM with a total lipid fraction derived from M. avium serovar 4 resulted in a significant suppression of lymphoproliferative responsiveness to phytohemagglutinin stimulation at concentrations not affecting cell viability. Although a similar suppression was not observed when PBM were treated with purified serovar 4-specific glycopeptidolipids (GPL), treatment with the 1-lipid fragment derived from the GPL did result in a significant suppression of phytohemagglutinin responsiveness. Further studies on August 6, 2020 by guest http://iai.asm.org/ Downloaded from

The ,8-elimination procedure was used to obtain two major fragments of Mycobacterium avium glycopeptidolipid antigens. The lipopeptide fragment, not the oligosaccharide, diminished the mitogen-induced blastogenic response of spleen cells at concentrations lower than those which affected viability. Electron microscopy revealed an internalization of lipopeptide and disruption of intracellular organization.on July 31, 2020 by guest http://iai.asm.org/ Downloaded from