Characterization of Aeromonas salmonicida mutants with low-level resistance to multiple antibiotics

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Abstract
Aeromonas salmonicida mutants selected for low-level resistance to small-molecular-mass antibiotics occur at frequencies that suggest point mutations and exhibit pleiotropic effects such as a multiple low-level antibiotic resistance, changes in outer membrane protein profiles, and loss of major exoprotease activity. Multiple low-level resistance appeared as the result of decreased outer membrane permeability associated with a change from a 38.5- to a 37-kilodalton (kDa) outer membrane protein. This decreased outer membrane permeability was determined by rates of nitrocefin hydrolysis by periplasmic beta-lactamase activity. The findings described above were supported by isolation of revertant strains selected for regained exoprotease activity, which also lost multiple low-level resistance and possessed outer membrane protein profiles indistinguishable from those of the original parent strains. Exoprotease from parent and revertant strains was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major extracellular protein of approximately 69 kDa. No accumulation of a protein in this molecular mass range was observed in extracellular or periplasmic fractions from the mutants. The results suggested that exoprotease loss is not simply the result of an inability to export protease from the periplasm because of outer membrane protein changes, as has been reported for certain mutants of some other gram-negative bacteria. Also, several growth conditions were used, including some that have been reported to influence outer membrane protein expression and permeability in other enteric gram-negative bacteria. Although exoprotease expression in A. salmonicida was influenced by these conditions, no major outer membrane protein changes which would correspond to changes observed in the mutants were observed in parent strains.