Antibacterial action of colistin (polymyxin E) against Mycobacterium aurum

Abstract

Mycobacterium aurum was susceptible to the antibiotic colistin (polymyxin E), which had an MIC of 5 ,ug/ml and an apparent bactericidal effect at concentrations above 50 ,ug/ml. Treatment of actively growing cells with sublethal concentrations of colistin (15 ,Ig/ml) resulted in synchronized cell division once the antibiotic was removed. Under conditions of synchronized cell growth, one cycle of DNA replication lasted 120 min and one cycle of cell division lasted about 180 min. Although the antibiotic treatment during synchronization experiments did not produce apparent changes in the bacterial envelope, it was accompanied by the accumulation of a polysaccharide-like substance in the bacterial cytoplasm which gradually decreased after the removal of antibiotic and by an increase in the number of mesosomes at 3 h after antibiotic removal. This step was closely linked to the doubling time of bacteria. Lethal concentrations of colistin of 50 and 100 ,ug/ml, which caused about 90 and 99% cell death, respectively, produced significant cytoplasmic membrane injuries, patchy appearance of the cell wall outer polysaccharide layer, and little cell lysis. These data indicate that the cytoplasmic membrane is a site of action of colistin and raise a question as to whether an outer bilayer exists in mycobacteria, at least functionally.The antibiotic colistin (polymyxin E) exerts its antibacterial action by interacting with membrane bilayers (la, 11, 21, 23). In gram-negative bacteria, which are surrounded by an outer membrane bilayer, the antibiotic causes characteristic deformities in the outer membrane, probably binding to lipopolysaccharides (7,8). Direct evidence of its interaction with the cytoplasmic membrane, however, has been more difficult to obtain (20). As condensation of the bacterial genome is an early event during the action of the antibiotic (20) and, moreover, the bacterial genome is attached to the plasma membrane (3, 18), which is probably necessary for its replication (6), we wondered whether treatment of cells with sublethal concentrations of colistin would result in a reversible detachment from the membrane. If this is the case, the removal of colistin would permit a simultaneous reattachment of the genome in all the cells, leading to synchronized cell division.To examine the mode of action of colistin and to test the above hypothesis about synchronized cell division, we selected Mycobacterium aurum, as it is susceptible to low concentrations of colistin (2). Like that of other mycobacteria (12)(13)(14)(15)(16)19), the cytoplasmic membrane of M. aurum has an asymmetrical profile, only the outer layer of which is specifically stained with the Thidry cytochemical method for electron microscopy (13,19). This observation facilitated the direct examination of the cytoplasmic membrane as a possible site of action of colistin. Moreover, M. aurum forms a polysaccharide outer layer (POL) which is homogenously distributed on the bacterial cell surface and can be revealed by ruthenium red (RR) staining (lla). Recen...

Rapid Killing of Acinetobacter baumannii by Polymyxins Is Mediated by a Hydroxyl Radical Death Pathway

“…However, there exist historical reports of polymyxins killing bacteria without actively lysing those cells (8,21). Here, we demonstrated that treatment with polymyxins induces hydroxyl radical production through the Fenton reaction and that this radical production mediates the rapid killing of A. baumannii, as well as E. coli and F. novicida.…”

“…It has often been assumed that these membrane disruptions cause bacterial cell death directly through membrane lysis. However, reports from as far back as the late 1970s indicate that under certain conditions, polymyxins are capable of killing bacteria without lysis, suggesting that another mechanism of bacterial cell death may also be induced by treatment with these antibiotics (8,21).…”

The antimicrobial effect of colistin methanesulfonate on Mycobacterium tuberculosis in vitro

“…Disruption of the hydrophobic barrier can allow for an influx of INH, leading to enhanced bactericidal activity. Another possibly is that during the self-promoted uptake, INH has an enhanced uptake from the environment by the cells, once again leading to an influx within the cell and enhanced bactericidal killing [18]. The Fenton reaction [46] should be investigated an additional mechanism of action against MTB.…”

“…The authors [51] observed no enhancement of drug activity for CST with RIF or INH, but no antagonism was observed either. colistin interacts with the outer polysaccharide layer, causing it to be patchy, more election dense with a budding appearance [18].…”

“…After 24 hours, cells suspensions (1 ml) were removed and centrifuged at 10 000 g"s for one minute. The supernatant was then removed, and the pellet was fixed for one hour at room temperature using 2.5 % v/v formaldehyde and glutaraldehyde in 0.1 M PIPES (Merck) (pH 7 -7.2), supplemented with 5 mM CaCl 2, MgCl 2 and 0.1 % w/v ruthenium red (EMD Millipore) [18]. Pellets were then rinsed using PIPES buffer (3 x ten minutes) followed by fixation in 1 % w/v OsO 4 (aq) (SPI Supplies, Philadelphia, USA) and rinsing with PIPES as described.…”