| Study on the in-vitro activities of aminoglycosides against Mycobacterium kansasii, Mycobacterium avium complex, and Mycobacterium fortuitum by the macrobroth dilution method showed that these non-tuberculous mycobacteria were resistant to multiple aminoglycosides; except amikacin which was active against both M. kansasii and M. fortuitum.;When screened for the presence of aminoglycoside-modifying enzymes (AMEs), aminoglycoside-acetyltransferase was detected in M. kansasii and M. fortuitum, but not in M. avium complex, while aminoglycoside-phosphotransferase and aminoglycoside-nucleotidyltransferase were absent from all three species tested. Aminoglycoside resistance in M. avium complex was, hence, not conferred by AMEs. Acetyltransferases from the standard strains of M. kansasii and M. fortuitum displayed relatively high Kms, all at the millimolar level, for substrates including tobramycin, neomycin, and kanamycin A. The Km of each substrate was well above the corresponding maximum achievable level in serum. The low affinities of these enzymes for their substrates suggested that drug modification in vivo was very unlikely. Among the various substrates tested, no apparent positive correlation was found between substrate affinity and resistance level. The presence of AMEs in these two mycobacterial species was therefore not shown to confer resistance to aminoglycosides.;Accumulation studies with radiolabelled amikacin showed that aminoglycoside accumulation in mycobacteria was firstly by diffusion through the cell wall barrier, followed by an electron-transport chain dependent process which could either be facilitated diffusion or active transport through the cytoplasmic membrane. Such uptake process was driven by a membrane potential and was dependent on the sizes, charges, and hydrophilicity of the drug molecules. Of the three species studied, M. avium complex was found to have the least permeable cell envelope, followed by M. kansasii and M. fortuitum. Reduced accumulation of amikacin, the most active drug among all aminoglycosides tested, confirmed that the broad-spectrum aminoglycoside resistance displayed by M. avium complex isolates was conferred by reduced drug accumulation. As for M. kansasii and M. fortuitum, their lack of any streptomycin-modifying enzyme and their significantly reduced streptomycin accumulation confirmed that reduced drug influx was also responsible for their intrinsic streptomycin resistance.;Though not very frequent, amikacin resistant strains of M. kansasii and M. fortuitum were found in this study. The absence of any significantly reduced amikacin accumulation and the lack of any amikacin-modifying enzyme in these resistant strains indicated that amikacin resistance in them was most likely due to target site(s) modification(s). Sequencing of the 1400 regions of their rrs genes confirmed that the molecular mechanism of amikacin resistance in the clinical isolate M. kansasii K4 was due to an A 1400 G mutation in its rrs gene. Such mutation, however, was absent in the other amikacin resistant clinical isolate M. fortuitum F32, suggesting that some other molecular mechanism(s) might be responsible. In both resistant strains, ribosomal resistance to amikacin probably did not result from abolishing the inhibition of protein synthesis as no reduced in-vivo binding of radiolabelled amikacin to their ribosomes in comparison with their susceptible counterparts was detected. Rather, it was due to restriction of the amikacin-induced misreading caused by target site(s) mutation(s). |
